胶质细胞谷氨酸转运体GLT-1表达上调及氨基酸平衡的维持参与大鼠脑缺血预处理的脑保护作用
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摘要
实验发现,给动物突然造成较严重的脑缺血,海马CA1区的神经元会大量死亡。若在此之前,预先给动物造成轻微、短时、不至于引起神经元死亡的脑缺血,可保护神经元,使其能够耐受在该轻微脑缺血后给予的通常会引起神经元严重损伤的较严重脑缺血。预先给予的轻微、短时脑缺血称为脑缺血预处理(cerebral ischemic preconditioning, CIP),所产生的这种保护作用称为脑缺血耐受(brain ischemic tolerance, BIT)。自1990年Kitagawa首先发现这一现象以来,大量研究证实了它的存在。阐明CIP脑保护作用的机制,对临床上研究、开发提高神经元对缺血缺氧耐受性的治疗方法具有重要意义。
大量研究表明,严重脑缺血缺氧可导致脑内谷氨酸等兴奋性氨基酸(excitatory amino acids, EAAs)的浓度异常升高,从而产生兴奋性神经毒作用。细胞膜上的高亲和性兴奋性氨基酸转运体(excitatory amino acid transporters, EAATs)可将兴奋性氨基酸从突触间隙转运至细胞内,在及时终止兴奋性突触传递以及维持细胞外液兴奋性氨基酸的正常水平中发挥重要作用。目前已发现五种高亲和性EAATs,包括EAAT1 (又称为glutamate/aspartate transporter, GLAST)、EAAT2 (又称为glail glutamate transporter-1, GLT-1)、EAAT3 (又称为excitatory amino acid carrier 1, EAAC1)、EAAT4和EAAT5。一般认为,GLAST和GLT-1为胶质细胞转运体,主要分布在星型胶质细胞;EAAC1、EAAT4和EAAT5为神经元转运体,其中EAAC1主要分布在海马神经元,EAAT4主要分布在小脑神经元,EAAT5主要分布在视网膜。EAATs主要由钠、钾离子浓度梯度驱动转运。当离子梯度降低或膜电位降低时,如缺血、癫痫发作时,EAATs摄取EAAs的功能减弱,甚至可以将细胞内的谷氨酸反向转运至细胞外,导致细胞外谷氨酸浓度异常升高,而产生神经毒作用。
尽管神经元和胶质细胞都表达EAATs,但是普遍认为胶质细胞的EAATs对谷氨酸的转运能力比神经元要强大的多,尤其是星形胶质细胞谷氨酸转运体亚型GLT-1在调节细胞外液谷氨酸浓度方面发挥主要作用。据此,我们推测,GLT-1可能在脑缺血耐受诱导过程中发挥作用。然而,到目前为止,仅有的几篇关于GLT-1是否在脑缺血耐受诱导过程中发挥作用的研究均为离体实验,且所得结果互相矛盾。为了探讨GLT-1是否在脑缺血耐受诱导过程中发挥保护作用,本实验应用脑组织病理学、免疫组织化学、western blot分析、脑内微透析以及高效液相色谱等方法,研究了在体(in vivo)脑缺血耐受诱导过程中大鼠海马GLT-1和胶质纤维酸性蛋白(Glial fibrillary acidic protein, GFAP)的表达以及谷氨酸、门冬氨酸、甘氨酸以和γ-氨基丁酸(γ-Aminobutyric acid, GABA)浓度的变化。
1 CIP诱导大鼠海马CA1区GLT-1和GFAP蛋白表达上调
采用大鼠四血管闭塞(4 vessel occlusion, 4VO)全脑缺血模型,应用脑组织病理学评价、免疫组织化学以及western blot分析等方法,观察脑缺血耐受诱导过程中大鼠海马CA1区GLT-1和GFAP蛋白表达的变化,探讨GLT-1在脑缺血耐受诱导过程中的作用。
1.1脑组织病理学评价
145只大鼠随机分为以下5组。①正常对照组(n=5);②凝闭椎动脉组(n=35):凝闭双侧椎动脉;③CIP组(n=35):全脑缺血3 min;④损伤性脑缺血组(n=35):全脑缺血8 min;⑤CIP+损伤性脑缺血组(n=35):CIP后2天全脑缺血8 min。除正常对照组之外,其余各组根据动物末次手术后取材时间,分为0 h(即刻)、3 h、1 d、2 d、3 d、5 d、7 d共7个亚组(每个时间点n=5)。各组动物均于预定时间点取脑,连续切片,硫堇染色下观察海马CA1区迟发性神经元死亡(delayed neuronal death, DND)情况。根据以下标准确定组织学分级(Histological grade, HG):0级,无神经元死亡;Ⅰ级,散在神经元死亡;Ⅱ级,成片神经元死亡;Ⅲ级,几乎全部的神经元死亡。计数海马CA1区每1 mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体神经元的数目,每张切片双侧海马各计数3个区段,取平均数为神经元密度(Neuronal density, ND)。
硫堇染色显示,正常对照组大鼠海马CA1区锥体神经元排列整齐致密,可见2～3层,细胞形态完整、边界清晰、尼氏小体丰富,胞核大而圆、核仁清晰, HG为0级, ND为210±5.7。凝闭椎动脉组大鼠各时间点海马CA1区均未见明显损伤,与control组相比,HG、ND均无明显变化。CIP组大鼠在各时间点海马CA1区均未见明显损伤,与凝闭椎动脉组大鼠相应时间点相比,HG、ND均无明显变化。损伤性脑缺血组大鼠在2天内未见明显的锥体神经元损伤;3天时可见部分神经元死亡,细胞形态发生明显改变,可见胞体缩小,形态不规则,呈多角型或梭型,胞膜皱缩,胞核固缩、浓染,核仁模糊不清或消失,突起明显深染变长;至损伤性脑缺血后5 d和7 d时,神经元几乎全部死亡,与凝闭椎动脉组大鼠相比,HG明显升高,ND明显减少。CIP+损伤性脑缺血组大鼠在各时间点海马CA1区锥体细胞排列整齐致密,胞核饱满,核仁较清晰,仅个别锥体细胞胞核固缩,无明显细胞缺失,与损伤性脑缺血后7 d组相比,HG明显降低,ND明显升高(p<0.01)。这些结果表明,CIP对2天后发生的严重脑缺血再灌注损伤有明显的对抗作用,表明脑缺血耐受诱导成功。
1.2免疫组织化学检测
动物分组及实验程序与上述脑组织病理学评价相同。
GLT-1免疫组织化学染色显示,对照组可见在海马CA1区有一定量GLT-1阳性标记物。凝闭椎动脉组几乎各时间点均可以见到GLT-1明显上调,以3h点表达最高,并且隐约可以见到胶质细胞样轮廓。CIP组与凝闭椎动脉组各相应时间点比较,早期明显上调,以1d时较为明显;另外可见在环绕锥体细胞的周围区域,GLT-1表达有所上调,形成一定的“网格”状。与凝闭椎动脉组相比,损伤性脑缺血组各相应时间点GLT-1的表达均下调,尤其以死亡的锥体细胞周围下调的最为明显,甚至表现为GLT-1大片缺失。CIP+损伤性脑缺血组大鼠与损伤性缺血组相比,各相应时间点GLT-1均明显上调,GLT-1阳性标记物紧紧包绕在锥体细胞周围,并形成明显的“网格”状。
GFAP免疫组化染色显示形态完整的星形胶质细胞呈星形或蜘蛛状,有明显的突起。control组海马CA1区可见星形胶质细胞散在均匀分布。凝闭椎动脉组与control组相比,所有时间点GFAP阳性标记物均明显减少。CIP与凝闭椎动脉组相比,CA1区星形胶质细胞的突起有所延长,在环绕锥体细胞的周围区域可见一定程度的GFAP免疫颗粒分布;GFAP阳性细胞数、总面积、平均光密度均明显上调(P<0.01),以1~3天较为明显。损伤性脑缺血组海马CA1区GFAP阳性细胞数目明显增多,胞体肥大,突起变长、增粗,但并不包绕锥体神经元胞体;GFAP阳性细胞数、总面积、平均光密度均显著升高(P<0.01);上述变化在损伤性脑缺血后3 d和5 d最为明显。CIP+损伤性脑缺血组,海马CA1区GFAP阳性细胞胞体并不明显增大,但突起明显延长并环绕锥体神经元胞体,形成非常明显的网格状;上述变化在损伤性脑缺血后2 d达高峰,一直持续至7 d。
1.3 western blot分析
205只大鼠随机分为以下6组:①正常对照组(n=5);②sham组(n=20):根据sham手术后取材时间,分为0 h(即刻)、3 h、12h、2d共4个时间点(每个时间点n=5);③椎动脉凝闭组(n=45);④CIP组(n=45);⑤损伤性脑缺血组(n=45);⑥CIP+损伤性脑缺血组(n=45)。除正常对照组及sham组之外,其余各组根据动物末次手术后的取材时间,分为0 h(即刻)、3 h、6h、12h、1 d、2 d、3 d、5 d、7 d共9个亚组(每个时间点n=5)。Sham组动物暴露椎动脉和颈总动脉,但不阻断其血流。其余各组动物的处理与1.1相同。
GLT-1的western blot分析显示,与对照组相比,凝闭椎动脉组GLT-1的表达在多个时间点明显上调,第一次高峰在3 h和6 h,第二次高峰在5 d,以3 h最高。与sham组相比,凝闭椎动脉组GLT-1的表达在即刻、3 h、2 d均显著升高,表明凝闭椎动脉后GLT-1的表达上调不是麻醉和手术导致的,而是凝闭椎动脉所引起的。CIP组中,与CIP后即刻或凝闭椎动脉组相比,除CIP后3 h、3 d GLT-1的表达明显下调外,其余各时间点GLT-1的表达均显著上调(P<0.05)。损伤性脑缺血组中,与该组的即刻时间点、或凝闭椎动脉组相比,除损伤性缺血后12 h外,其余所有时间点GLT-1的表达均下调。CIP+损伤性脑缺血组中,与该组的即刻取材时间点相比,GLT-1的表达在3 h、12 h明显上调;与损伤性脑缺血组相比,除1 d之外,其余各时间点GLT-1的表达均显著上调。
GFAP的western blot分析显示,与对照组相比,凝闭椎动脉组GFAP的表达于各时间点均明显下调。与对照相比,sham组所有时间点GFAP的表达均未见明显的变化。凝闭椎动脉组与sham相比,GFAP的表达在即刻、12 h、2 d均显著减少,表明凝闭椎动脉后GFAP的表达下调不是麻醉和手术导致的,而是凝闭椎动脉所引起的。CIP组中,与该组的即刻时间点相比,CIP后GFAP的表达在12 h、7 d明显上调;与凝闭椎动脉组相比,CIP后GFAP的表达在所有时间点均明显升高。损伤性脑缺血组中,与该组的即刻时间点相比,损伤性脑缺血后GFAP蛋白的表达在3 h、1 d、2 d、3 d、5 d、7 d均明显上调;与凝闭椎动脉组相比,损伤性脑缺血后GFAP的表达除即刻时间点之外,其余所有时间点均明显升高。CIP+损伤性脑缺血组中,与该组的即刻时间点相比,GFAP蛋白的表达在3 h、2 d、5 d、7 d明显上调;与损伤性脑缺血组相比,CIP+损伤性缺血后GFAP的表达在即刻时间点明显升高,3 h、1 d、7 d明显降低,其于各时间点无明显差异。
以上结果表明:3 min CIP在对抗8 min缺血打击引起的DND的同时,可引起海马CA1区星形胶质细胞的突起延长、包绕锥体神经元,并且表达大量的GLT-1,提示CIP引起的GLT-1表达上调参与CIP的脑保护作用。
2大鼠脑缺血耐受诱导过程中海马CA3区及齿状回GLT-1和GFAP蛋白表达的变化
模型制备、动物分组、实验程序及实验方法与第一部分相同。采用神经元死亡率(死亡神经元数目与该区域神经元总数目之比)表示CA3区及齿状回的DND。
2.1神经病理学评价
与海马CA1区不同,全脑缺血打击后,仅个别动物海马CA3区和齿状回出现了少量锥体细胞或颗粒细胞的DND,细胞缺失率不超过20%。预先给予CIP,同样可防止上述DND的发生。其余各组的变化与CA1相同。由此可见,海马CA3区和齿状回的神经元对缺血的耐受能力较强,CIP对这些神经元同样具有保护作用。
2.2 GLT-1和GFAP蛋白的表达
海马CA3区及齿状回GLT-1、GFAP蛋白的表达与海马CA1区有所不同。在正常对照组海马CA3区和齿状回,特别是CA3区即可见较多的星型胶质细胞的突起伸入锥体神经元层,并且围绕在锥体神经元周围,同时在锥体神经元之间的区域可以见到一定量的GLT-1免疫阳性颗粒;单纯凝闭椎动脉,即可使这些免疫阳性颗粒明显增多,形成了较为明显的“网格”现象;8 min全脑缺血打击后,大部分区域,特别是锥体神经元之间的部位的GLT-1和GFAP的表达进一步增强,使其所形成的锥体细胞层内的“网格”现象更为明显。但CA3及齿状回的个别神经元缺失区的GLT-1和GFAP的表达下降;单纯CIP以及CIP+脑缺血打击后,海马CA3区和齿状回GLT-1和GFAP的表达与CA1区相类似,但GLT-1和GFAP表达的上调及其所形成的“网格”现象更为明显。这些现象提示,海马CA3区和齿状回GLT-1的基础表达较高,并且受到缺血刺激时,GLT-1的反应性表达上调更显著。这些特点可能是海马CA3区和齿状回对缺血耐受性较强的原因之一。
3大鼠脑缺血耐受诱导过程中海马CA1区氨基酸浓度的变化
应用清醒动物脑内微透析和高效液相色谱技术,分析大鼠脑缺血耐受诱导过程中海马CA1区细胞外液中谷氨酸、门冬氨酸、甘氨酸和γ-氨基丁酸(GABA)浓度的变化,探讨氨基酸平衡的变化在脑缺血耐受诱导中的作用。24只大鼠随机分为4组(n=6):①凝闭椎动脉组;②CIP组;③损伤性脑缺血组;④CIP+损伤性脑缺血组。各组模型的制备与1.1相同。各组动物均在清醒状态下行背侧海马CA1区微透析。按预定程序收集透析液,高效液相色谱法测定透析液中上述氨基酸的浓度。各组动物于微透析结束后,常规饲养7天,断头取材行脑组织病理学评价,确定微透析探头的位置和海马CA1区组织病理学改变。
脑组织病理学观察显示,微透析探头被准确地植入了每例大鼠的背侧海马CA1区。除8 min脑缺血打击引起了海马CA1区几乎全部锥体神经元死亡之外,其余各组均未见到明显的神经元损伤。上述结果与我们以前的研究结果相一致。
在VAO组的所有透析标本之间,未观察到谷氨酸、门冬氨酸、甘氨酸及GABA浓度的明显变化。3分钟的全脑缺血引起谷氨酸、门冬氨酸、甘氨酸及GABA浓度迅速升高,分别达到其正常对照水平的1.5、2、1.9和2.3倍,随着血液再灌注,其浓度迅速降至正常水平。8分钟的全脑缺血引起谷氨酸、门冬氨酸、甘氨酸和GABA浓度迅速升高。甘氨酸和GABA的升高呈单峰、出现于脑缺血打击末,峰值为其正常对照水平的3~4倍,再灌注后很快恢复至对照水平。而谷氨酸和门冬氨酸的升高呈双峰状,其第一个高峰出现的时相及幅度与甘氨酸和GABA相似;第二个峰分别出现于再灌注后7分钟和19分钟,其峰值较第一个峰更高,分别为其正常对照水平的5~7倍。CIP+损伤性脑缺血组中,谷氨酸、门冬氨酸、甘氨酸及GABA的浓度呈单峰状升高,于损伤性脑缺血末达到峰顶,分别为其自身对照水平的1.7、2.5、7和4倍,再灌注后迅速降低至正常水平,并持续到实验结束。上述结果表明,缺血打击引起谷氨酸、门冬氨酸与GABA之间失衡;CIP可防止缺血打击所引起的谷氨酸、门冬氨酸与GABA之间的失衡,提示CIP所诱导的脑缺血耐受可能与其防止脑缺血打击引起的氨基酸失平衡有关。
此外,我们还对四血管闭塞法大鼠全脑缺血模型的制备进行了进一步探讨,发现凝闭双侧椎动脉本身也具有脑缺血预处理样作用,能够在一定程度上减轻其后48 h内较严重的全脑缺血所造成的损伤;椎动脉经寰椎的横突孔进入寰椎,行经寰椎内的翼状管,经翼状孔内口进入椎管。
4结论
(1) CIP引起大鼠海马CA1区星形胶质细胞的突起延长,伸入到锥体神经元之间并包绕锥体神经元,这些延长的突起上表达大量的GLT-1,此变化可以保护锥体神经元,使其能够耐受较严重的、通常会导致锥体神经元迟发性死亡的缺血打击。
(2)大鼠海马CA3区和齿状回的神经元对缺血的耐受能力较强,可能与该区GLT-1和GFAP的基础表达较高,以及受到缺血刺激时反应性表达上调更明显有关。这些发现进一步说明了GLT-1在BIT诱导中的作用。
(3)脑缺血打击引起大鼠海马CA1区谷氨酸、门冬氨酸与GABA之间失衡;CIP可通过防止缺血打击引起谷氨酸、门冬氨酸与GABA之间的失衡,从而诱导脑缺血耐受。
Transient sublethal cerebral ischemia could protect hippocampal neurons against delayed neuronal death (DND) induced normally by lethal ischemic insult. The transient cerebral ischemia is usually referred to as cerebral ischemic preconditioning (CIP), and the protective role is named as brain ischemic tolerance (BIT). The phenomenon was first found by Kitagawa in 1990 and proved by many other studies. It is very important to clearify the mechanisms of BIT induced by CIP for developing new therapeutic methods to enhance the tolerance of neurons to ischemia and hypoxia.
Many studies have proved that excitotoxicity of glutamate is an important mechanism for DND induced by transient global ischemic insult. Glutamate uptake is transiently reduced and the extracellular glutamate is increased after hypoxia-ischemia insults in the brain. Excitatory amino acid transporters (EAATs) are essential for maintaining normal extracellular level of glutamate. Five distinct high-affinity, sodium-dependent EAATs are identified in the rat brain, which include EAAT1 (glutamate/aspartate transporter, GLAST), EAAT2 (glial glutamate transpor-1, GLT-1), EAAT3 (excitatory amino acid carrier 1, EAAC1), EAAT4 and EAAT5. GLAST and GLT-1 are localized primarily in astrocytes. EAAC1 is widely distributed in hippocampal neurons. EAAT4 is localized mainly in cerebellar Purkinje cells. EAAT5 is mainly localized in retina. EAATs normally remove glutamate into cells and are driven by sodium, potassium and possibly by hydroxide ion gradients. Conversely, When the ion gradient or membrane potential drops, for instance during ischemia or epileptic activity, EAATs may decrease the uptake of glutamate, or even reverse and release glutamate into the extracellular space in a calcium-independent manner. Therefore, extracellular glutamate becomes abnormally high and leads to excitotoxicity.
Although both neurons and glia contain EAATs, it is generally accepted that the uptake capacity of astrocytes is much higher than that of neurons. Many studies have shown that GLT-1 plays a principal role in removing the released glutamate from the extracellular space and maintaining the extracellular glutamate below neurotoxic level in the brain. Considering the importance of GLT-1 in removing glutamate, it is reasonable to hypothesize that GLT-1 maybe play an important role in the acquisition of the brain ischemic tolerance induced by CIP. Unfortinately, there were limited reports until now concerning the role of GLT-1 in the induction of brain ischemic tolerance. Although they obtained a similar conclusion that GLT-1 participated in the induction of brain ischemic tolerance, the mechanisms underlying were just reversal. Additionally, they just performed the experiments in vitro. Little is known whether GLT-1 plays a role during the induction of brain ischemic tolerance in vivo. Therefore, the present study was undertaken to study whether GLT-1 participates in the induction of brain ischemic tolerance in vivo by observeing the expression of GLT-1 and glial fibrillary acidic protein (GFAP), a specific protein expressed by astrocytes, using immunohisto- chemistry and western blot analysis, and changes in concentrations of extracellular glutamate, aspartate, glycine andγ-aminobutyric acid (GABA) using brain microdialysis and high performance liquid chromatography (HPLC) during the induction of brain ischemic tolerance in rats.
1 The up-regulation of GLT-1 and GFAP in the rat hippocampal CA1 subfield induced by CIP
The rat global cerebral ischemic model was established by four-vessel occlusion. To clarify the role of GLT-1 during the induction of BIT in vivo, the expression of GLT-1 and GFAP in the CA1 hippocampus during the induction of brain ischemic tolerance in rats was observed by immunohistochemistry and western blotting.
1.1 Neuropathological evaluation
One hundred and forty five adult male Wistar rats were divided into 5 groups randomly:①control group (n=5);②vertebral artery occluding group (n=35): the bilateral vertebral arteries were electrocauterized permanently;③CIP group (n=35): a global brain ischemia for 3 min was given;④brain ischemic insult group (n=35): a global brain ischemic insult for 8 min was given;⑤CIP+ischemic insult group (n=35): a CIP was performed first and then a lethal global ischemic insult for 8 min was given 2 days after the CIP. The observations were performed at time 0 (immediate), 3 h, 1 d, 2 d, 3 d, 5 d and 7 d, after the last operation or treatment (n = 5 in each time point), except for the control group. At the determined endpoint of the experiment, the animals were sacrificed and the brain was removed for the neuropathological evaluation. The brain tissues were sectioned, and the delayed neuronal death (DND) was observed under staining with thionin. The histological changes of the hippocampal CA1 subfield were divided into 4 histological grade (HG) under the light microscope according to the following standard: grade 0, no neuron death; gradeⅠ, scattered single neuron death; gradeⅡ, mass neuron death; gradeⅢ, almost complete neuron death. The neuronal density (ND) of the hippocampal CA1 subfield was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1. The average of number of pyramidal neurons in 3 areas of the hippocampal CA1 subfield was calculated as value of ND.
Neuropathological evaluation showed that in the control rats, pyramidal neurons in the CA1 hippocampus were arranged in order with 2 to 3 cell layers, the outline of the neurons was intact, nucleus was full and nucleolus was clear. The HG was 0 and ND was 210±5.7 mm-1. No significant neuronal damage was observed in the CA1 subfield at all time points observed after VAO. Neither the HG nor the ND was different from that of the control group. No change in ND or HG was found at all time points after CIP compared with that of the control or VAO group. During the first two days after the lethal ischemic insult for 8 min, no significant pyramidal neuronal damage was observed in the hippocampal CA1 subfield. However, obvious DND was observed from the third day after the ischemic insult, such as decrease in ND and increase in HG. The damage deteriorated with time manifested as pyknosis of cell bodies, karyopyknosis of the nucleus, disappearance of the nucleolus. Almost complete neurons died on the fifth and seventh day after the lethal ischemic insult, represented by more significant decrease in ND and increase in HG compared with that of the third day after the lethal ischemic insult. When the animals were pretreated with the CIP 2 days before the lethal ischemic insult, the above injured changes were prevented clearly, which indicated that the CIP protected the pyramidal neurons in the CA1 hippocampus against the DND induced normally by the lethal ischemic insult.
1.2 Immunohistochemistry assay
Animals, grouping and protocols of the experiment were the same as those in neuropathological evaluation.
There were very weak, but diffuse immunoparticles distributed in the peri-pyramidal neuronal structure of the hippocampal CA1 subfield in the control group. The staining pattern is consistent with other reports. Compared with the control group, the intensity of GLT-1 immunoreactivity was markedly increased at almost all time points and showed morphological characteristics of astrocytes in the VAO group. The intensity of GLT-1 immunoreactivity was further increased after the CIP compared with that of the VAO group. Very interestingly, some GLT-1 immunoreactive particles were observed in the area between the pyramidal neurons, which tightly surrounded pyramidal neurons and made the pyramidal layer looked like“shaped grade”. Compared with the VAO group, the GLT-1 expression was markedly decreased at all time points after the lethal ischemic insult for 8 min, especially in the area where almost complete pyramidal neurons died, and the neighboring area of the pyramidal layer, even appeared as a sheet absence of GLT-1 immunoreactivity. But when the animals were pretreated with the CIP 2 days before the lethal ischemic insult, the decrease of the GLT-1 immunoreactivity induced by the lethal ischemic insult was prevented thoroughly. Moreover, there were more GLT-1 immunoreactive particles tightly surrounded the pyramidal neurons, which made the“shaped grid”observed in the CIP group to be more clear.
In the control group, GFAP immunostaining showed that astrocytes took on star or spider-like shape with prominent processes. Very few GFAP immunoreactive particles were observed in the area between the pyramidal neurons. Compared with the control group, significant down-regulation of GFAP immunoreactivity was observed at all time points in the VAO group, and few GFAP immunoreactive particles were observed in the area tightly surrounded the pyramidal neurons. After a CIP for 3 min, The GFAP expression was significantly up-regulated at almost all time points compared with that of the VAO group. Some GFAP immunoreactive particles were observed, like GLT-1, in the area between the pyramidal neurons, which tightly surrounded the pyramidal neurons and made the pyramidal layer look like“shaped grade”. The characteristics of immunostaining were similar with those of GLT-1 immunostaining mentioned above. Moreover, both the total area and average optical density of GFAP immunostaining after the CIP were significantly up-regulated compared with those of the VAO group. After the lethal brain ischemic insult for 8 min, astrocytes hypertrophied in soma with thickened processes and more intensive staining, which reached peak on 3 d after the lethal ischemia. However, no GFAP immunoreactive particles were observed in the area between the pyramidal neurons or the neighboring area of the pyramidal layer at all. From the fifth day after the lethal ischemic insult, the body of the astrocytes became more hypertrophic, whereas the processes of the hypertrophic astrocytes became collapsing and fragmenting. Although the number, total area and average optical density of immunoreactive cells were increased significantly after the lethal ischemic insult compared with those of the VAO group. When the animals were pretreated with a CIP 2 days before the lethal ischemic insult, there were many GFAP immunoreactive particles tightly surrounded the pyramidal neurons thoroughly, which made the“shaped grid”observed in the CIP group to be more clear. The phenomenon reached peak on 2 d and constantly lasted to 7 d (the end of the observed period in the experiment). However, both the number and average optical density of the GFAP immunoreactive cells were significantly decreased compared with those of the ischemic insult group.
1.3 Western blotting analysis
Two hundred and five adult male Wistar rats were divided into 6 groups randomly:①control group (n=5);②sham group (n=20);③VAO group (n=45);④CIP group (n=45);⑤brain ischemic insult group (n=45);⑥CIP+brain ischemic insult group (n=45). The western blotting analysis in the sham group was performed at time 0 (immediate), 3 h, 12h and 2 d, while in the other groups was performed at time 0 (immediate), 3 h, 6h, 12h, 1 d, 2 d, 3 d, 5 d and 7 d, after the last operation or treatment (n = 5 in each time point). In our preliminary experiment, some changes in expression of GLT-1 and GFAP were observed after VAO. To clarify whether the changes were induced by surgical procedures or by VAO, the sham group was designed in western blotting analysis. Rats in the sham group were subjected to a sham operation consisting of exposing of bilateral vertebral artery and bilateral common carotid arteries, but neither vertebral arteries nor common carotid arteries were occluded. The protocols of the rats in the other groups were the same as that in part 1.1.
Compared with the control group, no difference of GLT-1 levels was found at each time point observed in the sham group. However, the levels of GLT-1 expression were significantly up-regulated in CA1 subfield at almost all time points in the VAO group compared with that of the control group. The above results indicated that the GLT-1 up-regulation in the VAO group was induced by the occluding of vertebral arteries itself other than the anesthesia or surgical procedures. In the CIP group, the GLT-1 level at immediate time point was much higher than that of the control group. On the base of the up-regulated level of GLT-1 at the immediate time point, the expression of GLT-1 was further up-regulated after CIP for 3 min. In the brain ischemic insult group, the GLT-1 expression was significantly down-regulated at almost all time points compared with that of the VAO group. When the animals were pretreated with a CIP for 3 min 2 days before the lethal ischemic insult, the down-regulation of GLT-1 induced by lethal brain ischemic insult was prevented thoroughly by the CIP.
No difference of GFAP levels was found at every time points in the sham group compared with that of the control group. However, compared with the control group, the GFAP levels were significantly down-regulated at all time points after the occluding of the vertebral arteries. The above indicated that the GFAP down-regulation in the VAO group was induced by the occluding of vertebral arteries itself other than anesthesia or surgical operation. Compared with the VAO group, the GFAP levels were significantly up-regulated at almost all time points except the immediate time point after CIP. Compared with the VAO group, the GFAP levels were significantly up-regulated at almost all time points except the immediate time point after lethal brain ischemic insult for 8 min. Compared with the ischemic insult group, the GFAP levels were significantly down-regulated on 5 d and 7 d, whereas up-regulated at time 0 and 6 h in CIP+brain ischemic insult group.
Summary These results indicated that the surrounding of pyramidal neurons by astrocytes and up-regulation of GLT-1 induced by CIP played an important role in the acquisition of the brain ischemic tolerance induced by CIP.
2 Changes in the expression of GLT-1 and GFAP in the hippocampal CA3 subfield and dentate gyrus during the induction of brain ischemic tolerance in rats
The preparation of the model, grouping, protocols and methods were the same as those in part 1, except for that the neuropathological evaluation was performed by calculating percentages of injured neurons in the CA3 and DG.
2.1 Neuropathological evaluation
Lethal ischemic insult for 8 min induced mild DND in the CA3 subfield and dentate gyrus (DG) in some rats. The rate of neuronal death was approximately 5% in the CA3 subfield and 19% in DG. The CIP 2 days before the lethal brain ischemic insult could also protect the neurons in the CA3 subfield and DG against the DND induced normally by the lethal ischemic insult. All the above indicated that the neurons in the CA3 subfield and DG were relatively tolerated to ischemia, and CIP could also provide protection to the neurons in the CA3 subfield and DG.
2.2 The expression of GLT-1 and GFAP
Some differences in the expression of GLT-1 and GFAP were found in the CA3 subfield and DG compared with the CA1 subfield in the rat hippocampus. Processes of astrocytes in the CA3 subfield and DG, especially in the CA3 subfield, extended into the area between the neurons even in the control rats, which tightly surrounded neurons and made the neuronal layer looked like“shaped grade”. At the same time, many GLT-1 immunoreactive particles were observed in the same area. In the VAO group, both GFAP and GLT-1 immunoreactive particles in the area between the neurons in CA3 subfield and DG were significantly up-regulated by the VAO, which tightly surrounded neurons and made the“shaped grade”observed in the control group to be clearer. In the brain ischemic insult group, the GLT-1 expression was significantly increased and the“shaped grade”became more clearly in large area of the CA3 subfield and DG after the ischemic insult for 8 min, except for the significant decrease in the area where neurons died, and the neighboring area of the dead neurons. Compared with the CA1 subfield, the up-regulation of the expression of GLT-1 and GFAP to CIP for 3 min was also observed in CA3 subfield and DG, whereas the level of the up-regulation in CA3 subfield and DG was much higher than that in the CA1 subfield. In the CIP+ischemic insult group, similar response was observed among different subfields in the hippocampus, and the“shaped grade”existed in the CA3 subfield was very clear.
Summary The tolerance of the neurons in the CA3 subfield and DG to ischemia insult maybe related to the relatively higher basal expression and stronger responsive upregulation of GLT-1 and GFAP to ischemic stimulation in the area between neurons in the both subfields. These findings further illustrated the involvement of GLT-1 in the induction of BIT.
3. Changes in concentrations of amino acids in the extracellular fluid in the rat CA1 hippocampus during the induction of BIT
To investigate the role of the balance between excitatory amino acids (EAAs) and inhibitory amino acid in the induction of BIT, the concentration of glutamate, aspartate, glycine and GABA was analyzed during the induction of BIT by microdialysis combined with HPLC. Twenty four rats were divided into four groups randomly: vertebral artery occluding (VAO) group (n = 6), CIP group (n = 6), brain ischemic insult (II) group (n = 6), and CIP + brain ischemic insult group (n = 6). The preparations of the model were the same as those in part 1. The extracellular fluid in the rat CA1 region of the dorsal hippocampus was collected by intracerebral microdialysis in conscious rats. The concentration of amino acids in the dialysate was detected by HPLC. All rats were sacrificed on 7th day after the microdialysis for checking the position of the microdialysis probe and neuropathological evaluation.
In all cases the microdialysis probe was correctly positioned in the CA1 subfield of the rat dorsal hippocampus. Obvious DND was observed after the brain ischemic insult for 8 min, while no obvious neuronal damage was observed in other groups. The results were consistent with our previous study.
No changes in the concentration of each amino acid analyzed were observed among all samples from the VAO rats. In the CIP group, CIP for 3 min caused a significant acute increase coincident in both concentrations and time course of glutamate, aspartate, glycine and GABA, in which the peak values of the concentration were about 1.5, 2, 1.9, and 2.3 fold of their own average control level, respectively. In the brain ischemic insult group, lethal ischemic insult for 8 min evoked a significant increase in glutamate, aspartate, glycine and GABA. The acute increase of glycine and GABA appeared mono-peak at the end of the lethal ischemic insult. The peak value was about 3~4 folds of the average control level of its own. The increase of glutamate and aspartate showed a double-peak pattern. The first peaks were coincident in time course and magnitude with those in glycine and GABA. While the second peaks, which were about 5~7 fold of the control level, were higher in magnitude and appeared respectively at 7 min and 19 min after the reperfusion. In the CIP+ brain ischemic insult group, when the animals were pretreated with the CIP 2 days before the lethal ischemic insult, a significant acute increase of glutamate, aspartate, glycine and GABA coincident in both concentrations and time course was observed, in which the peak values were about 1.7, 2.5, 7, and 4 fold of their own average control level, respectively. The second higher peaks in glutamate and aspartate normally induced by brain ischemic insult were completely inhibited by the CIP. The results have shown that the lethal global brain ischemic insult for 8 min caused the imbalance between the EAAs such as glutamate as well as aspartate and GABA, the inhibitory amino acid; Whereas, when the animals were pretreated with the CIP for 3 min 2 days before the lethal ischemic insult, the increase among glutamate, aspartate, and GABA was coincident and kept balance.
Summary The lethal brain ischemic insult for 8 min caused imbalance between glutamate as well as aspartate and GABA in the CA1 hippocampus, which especially manifested as a delayed but more obvious increase in the concentration of glutamate and aspartate. The imbalance could be prevented by a preceded CIP, which could protect the pyramidal neurons of the CA1 hippocampus from DND normally induced by lethal brain ischemia. These findings indicated that preventing of the imbalance between the glutamate as well as aspartate and GABA maybe one of mechanisms involved in the neuroopretection of CIP.
In addition, we found that the prior occlusion of the bilateral vertebral arteries during producing 4VO global cerebral ischemic model might play a protective effect like cerebral ischemic preconditioning that can protect to some extent pyramidal neurons of the hippocampus against severe ischemic insult induced by occlusion of bilateral common carotid arteries within 48 h. The vertebral artery enters into the atlas via the transverse foramina, travels through the external aperture of the alar foramina, and then passes through the internal aperture of the alar foramina before entering into the vertebral canal.
4 Conclusions
(1) The surrounding of pyramidal neurons by astrocytes and up- regulation of GLT-1 induced by CIP played an important role in the acquisition of the brain ischemic tolerance induced by CIP
(2) The tolerance of the neurons in the CA3 subfield and DG to ischemia insult maybe related to the relatively higher basal expression and stronger responsive upregulation of GLT-1 and GFAP to ischemic stimulation in the area between neurons in the areas. These findings further illustrated the involvement of GLT-1 in the induction of BIT.
(3) The lethal brain ischemic insult for 8 min caused imbalance between glutamate as well as aspartate and GABA in the CA1 hippocampus, which especially manifested as a delayed but more obvious increase in the concentration of glutamate and aspartate. The imbalance could be prevented by a preceded CIP, which could protect the pyramidal neurons of the CA1 hippocampus from DND normally induced by lethal brain ischemia. These findings indicated that preventing of the imbalance between the glutamate as well as aspartate and GABA maybe one of mechanisms involved in the neuroopretection of CIP.
大量研究表明,严重脑缺血缺氧可导致脑内谷氨酸等兴奋性氨基酸(excitatory amino acids, EAAs)的浓度异常升高,从而产生兴奋性神经毒作用。细胞膜上的高亲和性兴奋性氨基酸转运体(excitatory amino acid transporters, EAATs)可将兴奋性氨基酸从突触间隙转运至细胞内,在及时终止兴奋性突触传递以及维持细胞外液兴奋性氨基酸的正常水平中发挥重要作用。目前已发现五种高亲和性EAATs,包括EAAT1 (又称为glutamate/aspartate transporter, GLAST)、EAAT2 (又称为glail glutamate transporter-1, GLT-1)、EAAT3 (又称为excitatory amino acid carrier 1, EAAC1)、EAAT4和EAAT5。一般认为,GLAST和GLT-1为胶质细胞转运体,主要分布在星型胶质细胞;EAAC1、EAAT4和EAAT5为神经元转运体,其中EAAC1主要分布在海马神经元,EAAT4主要分布在小脑神经元,EAAT5主要分布在视网膜。EAATs主要由钠、钾离子浓度梯度驱动转运。当离子梯度降低或膜电位降低时,如缺血、癫痫发作时,EAATs摄取EAAs的功能减弱,甚至可以将细胞内的谷氨酸反向转运至细胞外,导致细胞外谷氨酸浓度异常升高,而产生神经毒作用。
尽管神经元和胶质细胞都表达EAATs,但是普遍认为胶质细胞的EAATs对谷氨酸的转运能力比神经元要强大的多,尤其是星形胶质细胞谷氨酸转运体亚型GLT-1在调节细胞外液谷氨酸浓度方面发挥主要作用。据此,我们推测,GLT-1可能在脑缺血耐受诱导过程中发挥作用。然而,到目前为止,仅有的几篇关于GLT-1是否在脑缺血耐受诱导过程中发挥作用的研究均为离体实验,且所得结果互相矛盾。为了探讨GLT-1是否在脑缺血耐受诱导过程中发挥保护作用,本实验应用脑组织病理学、免疫组织化学、western blot分析、脑内微透析以及高效液相色谱等方法,研究了在体(in vivo)脑缺血耐受诱导过程中大鼠海马GLT-1和胶质纤维酸性蛋白(Glial fibrillary acidic protein, GFAP)的表达以及谷氨酸、门冬氨酸、甘氨酸以和γ-氨基丁酸(γ-Aminobutyric acid, GABA)浓度的变化。
1 CIP诱导大鼠海马CA1区GLT-1和GFAP蛋白表达上调
采用大鼠四血管闭塞(4 vessel occlusion, 4VO)全脑缺血模型,应用脑组织病理学评价、免疫组织化学以及western blot分析等方法,观察脑缺血耐受诱导过程中大鼠海马CA1区GLT-1和GFAP蛋白表达的变化,探讨GLT-1在脑缺血耐受诱导过程中的作用。
1.1脑组织病理学评价
145只大鼠随机分为以下5组。①正常对照组(n=5);②凝闭椎动脉组(n=35):凝闭双侧椎动脉;③CIP组(n=35):全脑缺血3 min;④损伤性脑缺血组(n=35):全脑缺血8 min;⑤CIP+损伤性脑缺血组(n=35):CIP后2天全脑缺血8 min。除正常对照组之外,其余各组根据动物末次手术后取材时间,分为0 h(即刻)、3 h、1 d、2 d、3 d、5 d、7 d共7个亚组(每个时间点n=5)。各组动物均于预定时间点取脑,连续切片,硫堇染色下观察海马CA1区迟发性神经元死亡(delayed neuronal death, DND)情况。根据以下标准确定组织学分级(Histological grade, HG):0级,无神经元死亡;Ⅰ级,散在神经元死亡;Ⅱ级,成片神经元死亡;Ⅲ级,几乎全部的神经元死亡。计数海马CA1区每1 mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体神经元的数目,每张切片双侧海马各计数3个区段,取平均数为神经元密度(Neuronal density, ND)。
硫堇染色显示,正常对照组大鼠海马CA1区锥体神经元排列整齐致密,可见2～3层,细胞形态完整、边界清晰、尼氏小体丰富,胞核大而圆、核仁清晰, HG为0级, ND为210±5.7。凝闭椎动脉组大鼠各时间点海马CA1区均未见明显损伤,与control组相比,HG、ND均无明显变化。CIP组大鼠在各时间点海马CA1区均未见明显损伤,与凝闭椎动脉组大鼠相应时间点相比,HG、ND均无明显变化。损伤性脑缺血组大鼠在2天内未见明显的锥体神经元损伤;3天时可见部分神经元死亡,细胞形态发生明显改变,可见胞体缩小,形态不规则,呈多角型或梭型,胞膜皱缩,胞核固缩、浓染,核仁模糊不清或消失,突起明显深染变长;至损伤性脑缺血后5 d和7 d时,神经元几乎全部死亡,与凝闭椎动脉组大鼠相比,HG明显升高,ND明显减少。CIP+损伤性脑缺血组大鼠在各时间点海马CA1区锥体细胞排列整齐致密,胞核饱满,核仁较清晰,仅个别锥体细胞胞核固缩,无明显细胞缺失,与损伤性脑缺血后7 d组相比,HG明显降低,ND明显升高(p<0.01)。这些结果表明,CIP对2天后发生的严重脑缺血再灌注损伤有明显的对抗作用,表明脑缺血耐受诱导成功。
1.2免疫组织化学检测
动物分组及实验程序与上述脑组织病理学评价相同。
GLT-1免疫组织化学染色显示,对照组可见在海马CA1区有一定量GLT-1阳性标记物。凝闭椎动脉组几乎各时间点均可以见到GLT-1明显上调,以3h点表达最高,并且隐约可以见到胶质细胞样轮廓。CIP组与凝闭椎动脉组各相应时间点比较,早期明显上调,以1d时较为明显;另外可见在环绕锥体细胞的周围区域,GLT-1表达有所上调,形成一定的“网格”状。与凝闭椎动脉组相比,损伤性脑缺血组各相应时间点GLT-1的表达均下调,尤其以死亡的锥体细胞周围下调的最为明显,甚至表现为GLT-1大片缺失。CIP+损伤性脑缺血组大鼠与损伤性缺血组相比,各相应时间点GLT-1均明显上调,GLT-1阳性标记物紧紧包绕在锥体细胞周围,并形成明显的“网格”状。
GFAP免疫组化染色显示形态完整的星形胶质细胞呈星形或蜘蛛状,有明显的突起。control组海马CA1区可见星形胶质细胞散在均匀分布。凝闭椎动脉组与control组相比,所有时间点GFAP阳性标记物均明显减少。CIP与凝闭椎动脉组相比,CA1区星形胶质细胞的突起有所延长,在环绕锥体细胞的周围区域可见一定程度的GFAP免疫颗粒分布;GFAP阳性细胞数、总面积、平均光密度均明显上调(P<0.01),以1~3天较为明显。损伤性脑缺血组海马CA1区GFAP阳性细胞数目明显增多,胞体肥大,突起变长、增粗,但并不包绕锥体神经元胞体;GFAP阳性细胞数、总面积、平均光密度均显著升高(P<0.01);上述变化在损伤性脑缺血后3 d和5 d最为明显。CIP+损伤性脑缺血组,海马CA1区GFAP阳性细胞胞体并不明显增大,但突起明显延长并环绕锥体神经元胞体,形成非常明显的网格状;上述变化在损伤性脑缺血后2 d达高峰,一直持续至7 d。
1.3 western blot分析
205只大鼠随机分为以下6组:①正常对照组(n=5);②sham组(n=20):根据sham手术后取材时间,分为0 h(即刻)、3 h、12h、2d共4个时间点(每个时间点n=5);③椎动脉凝闭组(n=45);④CIP组(n=45);⑤损伤性脑缺血组(n=45);⑥CIP+损伤性脑缺血组(n=45)。除正常对照组及sham组之外,其余各组根据动物末次手术后的取材时间,分为0 h(即刻)、3 h、6h、12h、1 d、2 d、3 d、5 d、7 d共9个亚组(每个时间点n=5)。Sham组动物暴露椎动脉和颈总动脉,但不阻断其血流。其余各组动物的处理与1.1相同。
GLT-1的western blot分析显示,与对照组相比,凝闭椎动脉组GLT-1的表达在多个时间点明显上调,第一次高峰在3 h和6 h,第二次高峰在5 d,以3 h最高。与sham组相比,凝闭椎动脉组GLT-1的表达在即刻、3 h、2 d均显著升高,表明凝闭椎动脉后GLT-1的表达上调不是麻醉和手术导致的,而是凝闭椎动脉所引起的。CIP组中,与CIP后即刻或凝闭椎动脉组相比,除CIP后3 h、3 d GLT-1的表达明显下调外,其余各时间点GLT-1的表达均显著上调(P<0.05)。损伤性脑缺血组中,与该组的即刻时间点、或凝闭椎动脉组相比,除损伤性缺血后12 h外,其余所有时间点GLT-1的表达均下调。CIP+损伤性脑缺血组中,与该组的即刻取材时间点相比,GLT-1的表达在3 h、12 h明显上调;与损伤性脑缺血组相比,除1 d之外,其余各时间点GLT-1的表达均显著上调。
GFAP的western blot分析显示,与对照组相比,凝闭椎动脉组GFAP的表达于各时间点均明显下调。与对照相比,sham组所有时间点GFAP的表达均未见明显的变化。凝闭椎动脉组与sham相比,GFAP的表达在即刻、12 h、2 d均显著减少,表明凝闭椎动脉后GFAP的表达下调不是麻醉和手术导致的,而是凝闭椎动脉所引起的。CIP组中,与该组的即刻时间点相比,CIP后GFAP的表达在12 h、7 d明显上调;与凝闭椎动脉组相比,CIP后GFAP的表达在所有时间点均明显升高。损伤性脑缺血组中,与该组的即刻时间点相比,损伤性脑缺血后GFAP蛋白的表达在3 h、1 d、2 d、3 d、5 d、7 d均明显上调;与凝闭椎动脉组相比,损伤性脑缺血后GFAP的表达除即刻时间点之外,其余所有时间点均明显升高。CIP+损伤性脑缺血组中,与该组的即刻时间点相比,GFAP蛋白的表达在3 h、2 d、5 d、7 d明显上调;与损伤性脑缺血组相比,CIP+损伤性缺血后GFAP的表达在即刻时间点明显升高,3 h、1 d、7 d明显降低,其于各时间点无明显差异。
以上结果表明:3 min CIP在对抗8 min缺血打击引起的DND的同时,可引起海马CA1区星形胶质细胞的突起延长、包绕锥体神经元,并且表达大量的GLT-1,提示CIP引起的GLT-1表达上调参与CIP的脑保护作用。
2大鼠脑缺血耐受诱导过程中海马CA3区及齿状回GLT-1和GFAP蛋白表达的变化
模型制备、动物分组、实验程序及实验方法与第一部分相同。采用神经元死亡率(死亡神经元数目与该区域神经元总数目之比)表示CA3区及齿状回的DND。
2.1神经病理学评价
与海马CA1区不同,全脑缺血打击后,仅个别动物海马CA3区和齿状回出现了少量锥体细胞或颗粒细胞的DND,细胞缺失率不超过20%。预先给予CIP,同样可防止上述DND的发生。其余各组的变化与CA1相同。由此可见,海马CA3区和齿状回的神经元对缺血的耐受能力较强,CIP对这些神经元同样具有保护作用。
2.2 GLT-1和GFAP蛋白的表达
海马CA3区及齿状回GLT-1、GFAP蛋白的表达与海马CA1区有所不同。在正常对照组海马CA3区和齿状回,特别是CA3区即可见较多的星型胶质细胞的突起伸入锥体神经元层,并且围绕在锥体神经元周围,同时在锥体神经元之间的区域可以见到一定量的GLT-1免疫阳性颗粒;单纯凝闭椎动脉,即可使这些免疫阳性颗粒明显增多,形成了较为明显的“网格”现象;8 min全脑缺血打击后,大部分区域,特别是锥体神经元之间的部位的GLT-1和GFAP的表达进一步增强,使其所形成的锥体细胞层内的“网格”现象更为明显。但CA3及齿状回的个别神经元缺失区的GLT-1和GFAP的表达下降;单纯CIP以及CIP+脑缺血打击后,海马CA3区和齿状回GLT-1和GFAP的表达与CA1区相类似,但GLT-1和GFAP表达的上调及其所形成的“网格”现象更为明显。这些现象提示,海马CA3区和齿状回GLT-1的基础表达较高,并且受到缺血刺激时,GLT-1的反应性表达上调更显著。这些特点可能是海马CA3区和齿状回对缺血耐受性较强的原因之一。
3大鼠脑缺血耐受诱导过程中海马CA1区氨基酸浓度的变化
应用清醒动物脑内微透析和高效液相色谱技术,分析大鼠脑缺血耐受诱导过程中海马CA1区细胞外液中谷氨酸、门冬氨酸、甘氨酸和γ-氨基丁酸(GABA)浓度的变化,探讨氨基酸平衡的变化在脑缺血耐受诱导中的作用。24只大鼠随机分为4组(n=6):①凝闭椎动脉组;②CIP组;③损伤性脑缺血组;④CIP+损伤性脑缺血组。各组模型的制备与1.1相同。各组动物均在清醒状态下行背侧海马CA1区微透析。按预定程序收集透析液,高效液相色谱法测定透析液中上述氨基酸的浓度。各组动物于微透析结束后,常规饲养7天,断头取材行脑组织病理学评价,确定微透析探头的位置和海马CA1区组织病理学改变。
脑组织病理学观察显示,微透析探头被准确地植入了每例大鼠的背侧海马CA1区。除8 min脑缺血打击引起了海马CA1区几乎全部锥体神经元死亡之外,其余各组均未见到明显的神经元损伤。上述结果与我们以前的研究结果相一致。
在VAO组的所有透析标本之间,未观察到谷氨酸、门冬氨酸、甘氨酸及GABA浓度的明显变化。3分钟的全脑缺血引起谷氨酸、门冬氨酸、甘氨酸及GABA浓度迅速升高,分别达到其正常对照水平的1.5、2、1.9和2.3倍,随着血液再灌注,其浓度迅速降至正常水平。8分钟的全脑缺血引起谷氨酸、门冬氨酸、甘氨酸和GABA浓度迅速升高。甘氨酸和GABA的升高呈单峰、出现于脑缺血打击末,峰值为其正常对照水平的3~4倍,再灌注后很快恢复至对照水平。而谷氨酸和门冬氨酸的升高呈双峰状,其第一个高峰出现的时相及幅度与甘氨酸和GABA相似;第二个峰分别出现于再灌注后7分钟和19分钟,其峰值较第一个峰更高,分别为其正常对照水平的5~7倍。CIP+损伤性脑缺血组中,谷氨酸、门冬氨酸、甘氨酸及GABA的浓度呈单峰状升高,于损伤性脑缺血末达到峰顶,分别为其自身对照水平的1.7、2.5、7和4倍,再灌注后迅速降低至正常水平,并持续到实验结束。上述结果表明,缺血打击引起谷氨酸、门冬氨酸与GABA之间失衡;CIP可防止缺血打击所引起的谷氨酸、门冬氨酸与GABA之间的失衡,提示CIP所诱导的脑缺血耐受可能与其防止脑缺血打击引起的氨基酸失平衡有关。
此外,我们还对四血管闭塞法大鼠全脑缺血模型的制备进行了进一步探讨,发现凝闭双侧椎动脉本身也具有脑缺血预处理样作用,能够在一定程度上减轻其后48 h内较严重的全脑缺血所造成的损伤;椎动脉经寰椎的横突孔进入寰椎,行经寰椎内的翼状管,经翼状孔内口进入椎管。
4结论
(1) CIP引起大鼠海马CA1区星形胶质细胞的突起延长,伸入到锥体神经元之间并包绕锥体神经元,这些延长的突起上表达大量的GLT-1,此变化可以保护锥体神经元,使其能够耐受较严重的、通常会导致锥体神经元迟发性死亡的缺血打击。
(2)大鼠海马CA3区和齿状回的神经元对缺血的耐受能力较强,可能与该区GLT-1和GFAP的基础表达较高,以及受到缺血刺激时反应性表达上调更明显有关。这些发现进一步说明了GLT-1在BIT诱导中的作用。
(3)脑缺血打击引起大鼠海马CA1区谷氨酸、门冬氨酸与GABA之间失衡;CIP可通过防止缺血打击引起谷氨酸、门冬氨酸与GABA之间的失衡,从而诱导脑缺血耐受。
Transient sublethal cerebral ischemia could protect hippocampal neurons against delayed neuronal death (DND) induced normally by lethal ischemic insult. The transient cerebral ischemia is usually referred to as cerebral ischemic preconditioning (CIP), and the protective role is named as brain ischemic tolerance (BIT). The phenomenon was first found by Kitagawa in 1990 and proved by many other studies. It is very important to clearify the mechanisms of BIT induced by CIP for developing new therapeutic methods to enhance the tolerance of neurons to ischemia and hypoxia.
Many studies have proved that excitotoxicity of glutamate is an important mechanism for DND induced by transient global ischemic insult. Glutamate uptake is transiently reduced and the extracellular glutamate is increased after hypoxia-ischemia insults in the brain. Excitatory amino acid transporters (EAATs) are essential for maintaining normal extracellular level of glutamate. Five distinct high-affinity, sodium-dependent EAATs are identified in the rat brain, which include EAAT1 (glutamate/aspartate transporter, GLAST), EAAT2 (glial glutamate transpor-1, GLT-1), EAAT3 (excitatory amino acid carrier 1, EAAC1), EAAT4 and EAAT5. GLAST and GLT-1 are localized primarily in astrocytes. EAAC1 is widely distributed in hippocampal neurons. EAAT4 is localized mainly in cerebellar Purkinje cells. EAAT5 is mainly localized in retina. EAATs normally remove glutamate into cells and are driven by sodium, potassium and possibly by hydroxide ion gradients. Conversely, When the ion gradient or membrane potential drops, for instance during ischemia or epileptic activity, EAATs may decrease the uptake of glutamate, or even reverse and release glutamate into the extracellular space in a calcium-independent manner. Therefore, extracellular glutamate becomes abnormally high and leads to excitotoxicity.
Although both neurons and glia contain EAATs, it is generally accepted that the uptake capacity of astrocytes is much higher than that of neurons. Many studies have shown that GLT-1 plays a principal role in removing the released glutamate from the extracellular space and maintaining the extracellular glutamate below neurotoxic level in the brain. Considering the importance of GLT-1 in removing glutamate, it is reasonable to hypothesize that GLT-1 maybe play an important role in the acquisition of the brain ischemic tolerance induced by CIP. Unfortinately, there were limited reports until now concerning the role of GLT-1 in the induction of brain ischemic tolerance. Although they obtained a similar conclusion that GLT-1 participated in the induction of brain ischemic tolerance, the mechanisms underlying were just reversal. Additionally, they just performed the experiments in vitro. Little is known whether GLT-1 plays a role during the induction of brain ischemic tolerance in vivo. Therefore, the present study was undertaken to study whether GLT-1 participates in the induction of brain ischemic tolerance in vivo by observeing the expression of GLT-1 and glial fibrillary acidic protein (GFAP), a specific protein expressed by astrocytes, using immunohisto- chemistry and western blot analysis, and changes in concentrations of extracellular glutamate, aspartate, glycine andγ-aminobutyric acid (GABA) using brain microdialysis and high performance liquid chromatography (HPLC) during the induction of brain ischemic tolerance in rats.
1 The up-regulation of GLT-1 and GFAP in the rat hippocampal CA1 subfield induced by CIP
The rat global cerebral ischemic model was established by four-vessel occlusion. To clarify the role of GLT-1 during the induction of BIT in vivo, the expression of GLT-1 and GFAP in the CA1 hippocampus during the induction of brain ischemic tolerance in rats was observed by immunohistochemistry and western blotting.
1.1 Neuropathological evaluation
One hundred and forty five adult male Wistar rats were divided into 5 groups randomly:①control group (n=5);②vertebral artery occluding group (n=35): the bilateral vertebral arteries were electrocauterized permanently;③CIP group (n=35): a global brain ischemia for 3 min was given;④brain ischemic insult group (n=35): a global brain ischemic insult for 8 min was given;⑤CIP+ischemic insult group (n=35): a CIP was performed first and then a lethal global ischemic insult for 8 min was given 2 days after the CIP. The observations were performed at time 0 (immediate), 3 h, 1 d, 2 d, 3 d, 5 d and 7 d, after the last operation or treatment (n = 5 in each time point), except for the control group. At the determined endpoint of the experiment, the animals were sacrificed and the brain was removed for the neuropathological evaluation. The brain tissues were sectioned, and the delayed neuronal death (DND) was observed under staining with thionin. The histological changes of the hippocampal CA1 subfield were divided into 4 histological grade (HG) under the light microscope according to the following standard: grade 0, no neuron death; gradeⅠ, scattered single neuron death; gradeⅡ, mass neuron death; gradeⅢ, almost complete neuron death. The neuronal density (ND) of the hippocampal CA1 subfield was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1. The average of number of pyramidal neurons in 3 areas of the hippocampal CA1 subfield was calculated as value of ND.
Neuropathological evaluation showed that in the control rats, pyramidal neurons in the CA1 hippocampus were arranged in order with 2 to 3 cell layers, the outline of the neurons was intact, nucleus was full and nucleolus was clear. The HG was 0 and ND was 210±5.7 mm-1. No significant neuronal damage was observed in the CA1 subfield at all time points observed after VAO. Neither the HG nor the ND was different from that of the control group. No change in ND or HG was found at all time points after CIP compared with that of the control or VAO group. During the first two days after the lethal ischemic insult for 8 min, no significant pyramidal neuronal damage was observed in the hippocampal CA1 subfield. However, obvious DND was observed from the third day after the ischemic insult, such as decrease in ND and increase in HG. The damage deteriorated with time manifested as pyknosis of cell bodies, karyopyknosis of the nucleus, disappearance of the nucleolus. Almost complete neurons died on the fifth and seventh day after the lethal ischemic insult, represented by more significant decrease in ND and increase in HG compared with that of the third day after the lethal ischemic insult. When the animals were pretreated with the CIP 2 days before the lethal ischemic insult, the above injured changes were prevented clearly, which indicated that the CIP protected the pyramidal neurons in the CA1 hippocampus against the DND induced normally by the lethal ischemic insult.
1.2 Immunohistochemistry assay
Animals, grouping and protocols of the experiment were the same as those in neuropathological evaluation.
There were very weak, but diffuse immunoparticles distributed in the peri-pyramidal neuronal structure of the hippocampal CA1 subfield in the control group. The staining pattern is consistent with other reports. Compared with the control group, the intensity of GLT-1 immunoreactivity was markedly increased at almost all time points and showed morphological characteristics of astrocytes in the VAO group. The intensity of GLT-1 immunoreactivity was further increased after the CIP compared with that of the VAO group. Very interestingly, some GLT-1 immunoreactive particles were observed in the area between the pyramidal neurons, which tightly surrounded pyramidal neurons and made the pyramidal layer looked like“shaped grade”. Compared with the VAO group, the GLT-1 expression was markedly decreased at all time points after the lethal ischemic insult for 8 min, especially in the area where almost complete pyramidal neurons died, and the neighboring area of the pyramidal layer, even appeared as a sheet absence of GLT-1 immunoreactivity. But when the animals were pretreated with the CIP 2 days before the lethal ischemic insult, the decrease of the GLT-1 immunoreactivity induced by the lethal ischemic insult was prevented thoroughly. Moreover, there were more GLT-1 immunoreactive particles tightly surrounded the pyramidal neurons, which made the“shaped grid”observed in the CIP group to be more clear.
In the control group, GFAP immunostaining showed that astrocytes took on star or spider-like shape with prominent processes. Very few GFAP immunoreactive particles were observed in the area between the pyramidal neurons. Compared with the control group, significant down-regulation of GFAP immunoreactivity was observed at all time points in the VAO group, and few GFAP immunoreactive particles were observed in the area tightly surrounded the pyramidal neurons. After a CIP for 3 min, The GFAP expression was significantly up-regulated at almost all time points compared with that of the VAO group. Some GFAP immunoreactive particles were observed, like GLT-1, in the area between the pyramidal neurons, which tightly surrounded the pyramidal neurons and made the pyramidal layer look like“shaped grade”. The characteristics of immunostaining were similar with those of GLT-1 immunostaining mentioned above. Moreover, both the total area and average optical density of GFAP immunostaining after the CIP were significantly up-regulated compared with those of the VAO group. After the lethal brain ischemic insult for 8 min, astrocytes hypertrophied in soma with thickened processes and more intensive staining, which reached peak on 3 d after the lethal ischemia. However, no GFAP immunoreactive particles were observed in the area between the pyramidal neurons or the neighboring area of the pyramidal layer at all. From the fifth day after the lethal ischemic insult, the body of the astrocytes became more hypertrophic, whereas the processes of the hypertrophic astrocytes became collapsing and fragmenting. Although the number, total area and average optical density of immunoreactive cells were increased significantly after the lethal ischemic insult compared with those of the VAO group. When the animals were pretreated with a CIP 2 days before the lethal ischemic insult, there were many GFAP immunoreactive particles tightly surrounded the pyramidal neurons thoroughly, which made the“shaped grid”observed in the CIP group to be more clear. The phenomenon reached peak on 2 d and constantly lasted to 7 d (the end of the observed period in the experiment). However, both the number and average optical density of the GFAP immunoreactive cells were significantly decreased compared with those of the ischemic insult group.
1.3 Western blotting analysis
Two hundred and five adult male Wistar rats were divided into 6 groups randomly:①control group (n=5);②sham group (n=20);③VAO group (n=45);④CIP group (n=45);⑤brain ischemic insult group (n=45);⑥CIP+brain ischemic insult group (n=45). The western blotting analysis in the sham group was performed at time 0 (immediate), 3 h, 12h and 2 d, while in the other groups was performed at time 0 (immediate), 3 h, 6h, 12h, 1 d, 2 d, 3 d, 5 d and 7 d, after the last operation or treatment (n = 5 in each time point). In our preliminary experiment, some changes in expression of GLT-1 and GFAP were observed after VAO. To clarify whether the changes were induced by surgical procedures or by VAO, the sham group was designed in western blotting analysis. Rats in the sham group were subjected to a sham operation consisting of exposing of bilateral vertebral artery and bilateral common carotid arteries, but neither vertebral arteries nor common carotid arteries were occluded. The protocols of the rats in the other groups were the same as that in part 1.1.
Compared with the control group, no difference of GLT-1 levels was found at each time point observed in the sham group. However, the levels of GLT-1 expression were significantly up-regulated in CA1 subfield at almost all time points in the VAO group compared with that of the control group. The above results indicated that the GLT-1 up-regulation in the VAO group was induced by the occluding of vertebral arteries itself other than the anesthesia or surgical procedures. In the CIP group, the GLT-1 level at immediate time point was much higher than that of the control group. On the base of the up-regulated level of GLT-1 at the immediate time point, the expression of GLT-1 was further up-regulated after CIP for 3 min. In the brain ischemic insult group, the GLT-1 expression was significantly down-regulated at almost all time points compared with that of the VAO group. When the animals were pretreated with a CIP for 3 min 2 days before the lethal ischemic insult, the down-regulation of GLT-1 induced by lethal brain ischemic insult was prevented thoroughly by the CIP.
No difference of GFAP levels was found at every time points in the sham group compared with that of the control group. However, compared with the control group, the GFAP levels were significantly down-regulated at all time points after the occluding of the vertebral arteries. The above indicated that the GFAP down-regulation in the VAO group was induced by the occluding of vertebral arteries itself other than anesthesia or surgical operation. Compared with the VAO group, the GFAP levels were significantly up-regulated at almost all time points except the immediate time point after CIP. Compared with the VAO group, the GFAP levels were significantly up-regulated at almost all time points except the immediate time point after lethal brain ischemic insult for 8 min. Compared with the ischemic insult group, the GFAP levels were significantly down-regulated on 5 d and 7 d, whereas up-regulated at time 0 and 6 h in CIP+brain ischemic insult group.
Summary These results indicated that the surrounding of pyramidal neurons by astrocytes and up-regulation of GLT-1 induced by CIP played an important role in the acquisition of the brain ischemic tolerance induced by CIP.
2 Changes in the expression of GLT-1 and GFAP in the hippocampal CA3 subfield and dentate gyrus during the induction of brain ischemic tolerance in rats
The preparation of the model, grouping, protocols and methods were the same as those in part 1, except for that the neuropathological evaluation was performed by calculating percentages of injured neurons in the CA3 and DG.
2.1 Neuropathological evaluation
Lethal ischemic insult for 8 min induced mild DND in the CA3 subfield and dentate gyrus (DG) in some rats. The rate of neuronal death was approximately 5% in the CA3 subfield and 19% in DG. The CIP 2 days before the lethal brain ischemic insult could also protect the neurons in the CA3 subfield and DG against the DND induced normally by the lethal ischemic insult. All the above indicated that the neurons in the CA3 subfield and DG were relatively tolerated to ischemia, and CIP could also provide protection to the neurons in the CA3 subfield and DG.
2.2 The expression of GLT-1 and GFAP
Some differences in the expression of GLT-1 and GFAP were found in the CA3 subfield and DG compared with the CA1 subfield in the rat hippocampus. Processes of astrocytes in the CA3 subfield and DG, especially in the CA3 subfield, extended into the area between the neurons even in the control rats, which tightly surrounded neurons and made the neuronal layer looked like“shaped grade”. At the same time, many GLT-1 immunoreactive particles were observed in the same area. In the VAO group, both GFAP and GLT-1 immunoreactive particles in the area between the neurons in CA3 subfield and DG were significantly up-regulated by the VAO, which tightly surrounded neurons and made the“shaped grade”observed in the control group to be clearer. In the brain ischemic insult group, the GLT-1 expression was significantly increased and the“shaped grade”became more clearly in large area of the CA3 subfield and DG after the ischemic insult for 8 min, except for the significant decrease in the area where neurons died, and the neighboring area of the dead neurons. Compared with the CA1 subfield, the up-regulation of the expression of GLT-1 and GFAP to CIP for 3 min was also observed in CA3 subfield and DG, whereas the level of the up-regulation in CA3 subfield and DG was much higher than that in the CA1 subfield. In the CIP+ischemic insult group, similar response was observed among different subfields in the hippocampus, and the“shaped grade”existed in the CA3 subfield was very clear.
Summary The tolerance of the neurons in the CA3 subfield and DG to ischemia insult maybe related to the relatively higher basal expression and stronger responsive upregulation of GLT-1 and GFAP to ischemic stimulation in the area between neurons in the both subfields. These findings further illustrated the involvement of GLT-1 in the induction of BIT.
3. Changes in concentrations of amino acids in the extracellular fluid in the rat CA1 hippocampus during the induction of BIT
To investigate the role of the balance between excitatory amino acids (EAAs) and inhibitory amino acid in the induction of BIT, the concentration of glutamate, aspartate, glycine and GABA was analyzed during the induction of BIT by microdialysis combined with HPLC. Twenty four rats were divided into four groups randomly: vertebral artery occluding (VAO) group (n = 6), CIP group (n = 6), brain ischemic insult (II) group (n = 6), and CIP + brain ischemic insult group (n = 6). The preparations of the model were the same as those in part 1. The extracellular fluid in the rat CA1 region of the dorsal hippocampus was collected by intracerebral microdialysis in conscious rats. The concentration of amino acids in the dialysate was detected by HPLC. All rats were sacrificed on 7th day after the microdialysis for checking the position of the microdialysis probe and neuropathological evaluation.
In all cases the microdialysis probe was correctly positioned in the CA1 subfield of the rat dorsal hippocampus. Obvious DND was observed after the brain ischemic insult for 8 min, while no obvious neuronal damage was observed in other groups. The results were consistent with our previous study.
No changes in the concentration of each amino acid analyzed were observed among all samples from the VAO rats. In the CIP group, CIP for 3 min caused a significant acute increase coincident in both concentrations and time course of glutamate, aspartate, glycine and GABA, in which the peak values of the concentration were about 1.5, 2, 1.9, and 2.3 fold of their own average control level, respectively. In the brain ischemic insult group, lethal ischemic insult for 8 min evoked a significant increase in glutamate, aspartate, glycine and GABA. The acute increase of glycine and GABA appeared mono-peak at the end of the lethal ischemic insult. The peak value was about 3~4 folds of the average control level of its own. The increase of glutamate and aspartate showed a double-peak pattern. The first peaks were coincident in time course and magnitude with those in glycine and GABA. While the second peaks, which were about 5~7 fold of the control level, were higher in magnitude and appeared respectively at 7 min and 19 min after the reperfusion. In the CIP+ brain ischemic insult group, when the animals were pretreated with the CIP 2 days before the lethal ischemic insult, a significant acute increase of glutamate, aspartate, glycine and GABA coincident in both concentrations and time course was observed, in which the peak values were about 1.7, 2.5, 7, and 4 fold of their own average control level, respectively. The second higher peaks in glutamate and aspartate normally induced by brain ischemic insult were completely inhibited by the CIP. The results have shown that the lethal global brain ischemic insult for 8 min caused the imbalance between the EAAs such as glutamate as well as aspartate and GABA, the inhibitory amino acid; Whereas, when the animals were pretreated with the CIP for 3 min 2 days before the lethal ischemic insult, the increase among glutamate, aspartate, and GABA was coincident and kept balance.
Summary The lethal brain ischemic insult for 8 min caused imbalance between glutamate as well as aspartate and GABA in the CA1 hippocampus, which especially manifested as a delayed but more obvious increase in the concentration of glutamate and aspartate. The imbalance could be prevented by a preceded CIP, which could protect the pyramidal neurons of the CA1 hippocampus from DND normally induced by lethal brain ischemia. These findings indicated that preventing of the imbalance between the glutamate as well as aspartate and GABA maybe one of mechanisms involved in the neuroopretection of CIP.
In addition, we found that the prior occlusion of the bilateral vertebral arteries during producing 4VO global cerebral ischemic model might play a protective effect like cerebral ischemic preconditioning that can protect to some extent pyramidal neurons of the hippocampus against severe ischemic insult induced by occlusion of bilateral common carotid arteries within 48 h. The vertebral artery enters into the atlas via the transverse foramina, travels through the external aperture of the alar foramina, and then passes through the internal aperture of the alar foramina before entering into the vertebral canal.
4 Conclusions
(1) The surrounding of pyramidal neurons by astrocytes and up- regulation of GLT-1 induced by CIP played an important role in the acquisition of the brain ischemic tolerance induced by CIP
(2) The tolerance of the neurons in the CA3 subfield and DG to ischemia insult maybe related to the relatively higher basal expression and stronger responsive upregulation of GLT-1 and GFAP to ischemic stimulation in the area between neurons in the areas. These findings further illustrated the involvement of GLT-1 in the induction of BIT.
(3) The lethal brain ischemic insult for 8 min caused imbalance between glutamate as well as aspartate and GABA in the CA1 hippocampus, which especially manifested as a delayed but more obvious increase in the concentration of glutamate and aspartate. The imbalance could be prevented by a preceded CIP, which could protect the pyramidal neurons of the CA1 hippocampus from DND normally induced by lethal brain ischemia. These findings indicated that preventing of the imbalance between the glutamate as well as aspartate and GABA maybe one of mechanisms involved in the neuroopretection of CIP.
引文
1 Arriza JL, Eliasof S, Kavanaugh MP, et al Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc Natl Acad Sci USA, 1997, 94:4155~4160
2 Atochin DN, Clark J, Demchenko IT, et al Rapid cerebral ischemic preconditioning in mice deficient in endothelial and neuronal nitric oxide synthases. Stroke, 2003, 34(5):1299~1303
3 Chen J, Graham SH, Zhu RL, et al Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab, 1996, 16:566~577
4 Chen JC, Hcu-Chou H, Lu JL, et al Down-regulation of the glial glutamate transporter GLT-1 in rat hippocampus and striatum and its modulation by a group III metabotropic glutamate receptor antagonist following transient global forebrain ischemia. Neuropharmacology, 2005, 49(5):703~714
5 Fairman WA, Vandenberg RJ, Arriza JL, et al An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature, 1995, 375:599~603
6 Garcia L, Burda J, Hrehorovska M, et al Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression. J Neurochem, 2004, 88(1):136~147
7 Heurteaux C, Lauritzen I, Widmann C, et al Essential role of adenosine,adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning. PNAS, 1995, 92: 4666~4670
8 Kato H, Liu Y, Araki T, et al Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res, 1991, 553:238~242
9 Kato H, Kogure K, Biochemical and molecular characteristics of the brain with developing cerebral infarction. Cell Mol Neurobiol, 1999, 19:93~108
10 Kawahara K, Kosugi T, Tanaka M, et al Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning- induced ischemic tolerance of neurons in neuron/astrocyte co-cultures. Glia, 2005, 49:349~359
11 Kirino T, Ischemic tolerance. J Cereb Blood Flow Metab, 2002, 22:1283~2196
12 Kitagawa K, Matsumoto M, Tagaya M, et al ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res, 1990, 528:21~24
13 Kosugi T, Kawahara K, Yamada T,et al Functional significance of the preconditioning-induced down-regulation of glutamate transporter GLT-1 in neuron/astrocyte co-cultures. Neurochem res, 2005, 30(9):1109~1116
14 Lehre KP, Levy LM, Ottersen OP, et al Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocyto- chemical observations. J Neurosci, 1995, 3:1835~1853
15 Levy LM, Lehre KP, Rolstad B, et al A monoclonal antibody raised against an [Na+ -K+] coupled L-glutamate transporter purified from rat brain confirms glial cell localization. FEBS Lett, 1993, 317:79~84
16 Lipton P, Ischemic cell death in brain neurons. Physiol Rev, 1999, 79:1431~1568
17 Liu HQ, Li WB, Li SQ, et al Nitric oxide participates in the induction of brain ischemic tolerance via activating ERK1/2 signaling pathways. Neurochem Res, 2006, 31(7):967~974
18 Mitani A, Tanaka K. Functional Changes of Glial Glutamate transporterGLT-1 during Ischemia: An In Vivo Study in the Hippocampal CA1 of Normal Mice and Mutant Mice Lacking GLT-1. J Neurosci, 2003, 23(18):7176~7182
19 Neary JT, McCarthy M, Kang Y, et al Mitogenic signaling from P1 and P2 purinergic receptors to mitogen-activated protein kinase in human fetal astrocytes. Neurosci Letters, 1998, 242:159~162
20 Neary JT, Kang Y, Shi YF, et al P2 receptor signalling, proliferation of astrocytes, and expression of molecules involved in cell-cell interactions. Novartis Found Symp, 2006, 276:131-43; discussion 143-7, 233-7, 275-81
21 Nicotera P, Bano D. The enemy at the gates: Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell, 2003, 115(7):768~770
22 Nishino K, Nowak Jr TS. Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning. J Cereb Blood Flow Metab, 2004, 24(2):167~178
23 Payet O, Maurin L, Bonne C, et al Hypoxia stimulates glutamate uptake in whole rat retinal cells in vitro. Neurosci Lett, 2004, 356(2):148~150
24 Pines G, Danbolt NC, Bj?r?s M, et al Cloning and expression of a rat brain L-glutamate transporter. Nature, 1992, 360:464~467
25 Pulsinelli WA, Brierley JB. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke, 1979, 10(3):267~272
26 Raghavendra Rao VL, Rao AM, Dogen A, et al Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia. Neurochem Int, 2000, 36(6):531~537
27 Rao L, Dogan A, Todd KG, et al Antisense Knockdown of the Glial Glutamate Transporter GLT-1, But Not the Neuronal Glutamate Transporter EAAC1, Exacerbates Transient Focal Cerebral Ischemia-Induced Neuronal Damage in Rat Brain. J Neurosci, 2001, 21(6):1876~1883
28 Rao VL, Dogan A, Todd KG, et al Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain. J Neurosci, 2001a, 21(6):1876~1883
29 Rao VL, Dogan A, Bowen KK, et al Antisense knockdown of the glial glutamate transporter GLT-1 exacerbates hippocampal neuronal damage following traumatic injury to rat brain. Eur J Neurosci, 2001b, 13(1):119~128
30 Rauen T, Kanner BI. Localization of the glutamate transporter GLT-1 in rat and macaque monkey retinae. Neurosci Lett, 1994, 169:137~140
31 Romera C, Hurtado O, Botella SH, et al In vitro ischemic tolerance involves upregulation of glutamate transport partly mediated by the TACE/ADAM17-tumor necrosis factor-alpha pathway. J Neurosci, 2004,
24(6):1350~1357
32 Rossi DJ, Oshima T, Attwell D. Glutamate release in severe brain ischaemia is mainly by reversed uptake, Nature, 2000, 403:316~321
33 Rothstein JD, Martin L, Levey AI, et al Localization of neuronal and glial glutamate transporters. Neuron, 1994, 13:713~725
34 Rothstein JD, Dykes-Hoberg M, Pardo CA, et al Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron, 1996, 16:675~686
35 Seki Y, Feustel PJ, Keller RW Jr, et al Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker. Stroke, 1999, 30(2):433~440
36 Storck T, Schulte S, Hofmann K, et al Structure, expression, and functional analysis of a Na+-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci USA, 1992, 89:10955~10959
37 Tanaka K, Watase K, Manabe T, et al Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science, 1997, 276:1699~1702
38 Tomimoto H, Takemoto O, Akiguchi I, et al Immunoelectron microscopicstudy of C-fos, C-Jun and heat shock protein after transient cerebral ischemia in gerbils. Acta Neuropathol 1999, 97:22~30
39 Yamada K, Watanabe M, Shibata T, et al Glutamate transporter GLT-1 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression. J Neurosci, 1998, 18:5706~5713
40 Yeh TH, Hwang HM, Chee JJ, et al Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia. Neurobiol Dis, 2005, 18(3):476~483
41 Zacco A, Togo J, Spence K, et al 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitors protect cortical neurons from excitotoxicity. Neuroscience, 2003, 23(35):11104~11111
42 Zhou AM, Li WB, Li QJ, et al A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res, 2004, 48(4): 379~404
1 Benveniste H, Drejer J, Schousboe A, et al Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem, 1984, 43(5):1369~1374
2 Hossmann, KA, Thresholds of ischemic injury. In: Ginsberg, MD, Bogousslavsky, J (Eds.), Cerebrovascular Disease. Pathophysiology, Diagnosis, and Treatment, 1998, vol. 1. Blackwell Science, Malden, MA, USA, pp. 193~204
3 Liu S, Lau L, Wei J, et al Expression of Ca2+-permeable AMPA receptor channels primes cell death in transient forebrain ischemia. Neuron, 2004, 43: 43~55
4 Nicotera P, Bano D, The enemy at the gates: Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell, 2003, 115(7):768~770
5 Schmidt-Kastner R, Freund TF, Selective vulnerability of the hippocampus in brain ischemia. Neuroscience, 1991, 40: 599~636
6 Yeh TH, Hwang HM, Chee JJ, et al Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia. Neurobiol Dis, 2005, 18(3):476~483
1 Atochin DN, Clark J, Demchenko IT et al Rapid cerebral ischemicpreconditioning in mice deficient in endothelial and neuronal nitric oxide synthases. Stroke, 2003, 34(5):1299~1303
2 Attwell D, Barbour B, Szatkowski M, Nonvesicular release of neurotransmitter. Neuron, 1993, 11:401~407
3 Baker DA, Xi ZX, Shen H, et al The origin and neuronal function of in vivo nonsynaptic glutamate. J Neurosci, 2002, 22: 9134~9141
4 Benveniste H, The excitotoxin hypothesis in relation to cerebral ischemia, Cerebrovasc. Brain Metab Rev, 1991, 3:213~245
5 Bradford HF, Glutamate, GABA and epilepsy. Prog. Neurobiol, 1995, 47: 477~511
6 Bradford SE, Nadler JV, Aspartate release from rat hippocampal synaptosomes. Neuroscience, 2004, 128(4):751~65
7 Chatterton JE, Awobuluyi M, Premkumar LS, et al Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits. Nature, 2002, 415:793~798.
8 Chen J, Graham SH, Zhu RL, et al Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab, 1996, 16:566~77
9 Choi DW, Rothman SM, The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Rev Neurosci, 1990, 13:171~182
10 French ED, Vezzani A, Whetsell Jr WO, et al Antiexcitotoxic actions of taurine in the rat hippocampus studied in vivo and in vitro. Adv Exp Med Biol, 1986, 203:349~362
11 Garcia L, Burda J, Hrehorovska M, et al Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression. J Neurochem, 2004, 88(1):136~47
12 Gemba T, Matsunaga K, Ueda M, Changes in extracellular concentration of amino acids in the hippocampus during cerebral ischemia in stroke-prone SHR, stroke-resistant SHR and normotensive rats. Neurosci Lett, 1992, 135: 184~188
13 Hertz L, Zielke HR, Astrocytic control of glutamatergic activity:astrocytes as stars of the show. Trends Neurosci, 2004, 27:735~743
14 Kirino T, Ischemic tolerance. J Cereb Blood Flow Metab, 2002, 22:1283~1296
15 Hillered L, Hallstrom A, Segersvard S, et al Dynamics of extracellular metabolites in the striatum after middle cerebral artery occlusion in the rat monitored by intracerebral microdialysis. J Cereb Blood Flow Metab, 1989, 9:607~616
16 Huang YH, Bergles DE, Glutamate transporters bring competition to the synapse. Curr Opin Neurobiol, 2004, 14:346~352
17 Johansen EF, Diemer NH, Enhancement of GABA neurotransmission after cerebral ischemia in the rat reduces loss of hippocampal CA1 pyramidal cells. Acta Neurol Scand, 1991, 84:1~6
18 Katsuki H, Akaike A. Excitotoxic degeneration of hypothalamic orexin neurons in slice culture. Neurobiol Dis, 2004, 15(1):61~9
19 Liu HQ, Li WB, Li SQ, et al Nitric oxide participates in the induction of brain ischemic tolerance via activating ERK1/2 signaling pathways. Neurochem Res, 2006, 31(7):967~974
20 Kato H, Liu Y, Araki T, et al Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage followingsecondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res, 1991, 553:238~242
21 Kawahara K, Kosugi T, Tanaka M, et al Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning- induced ischemic tolerance of neurons in neuron/ astrocyte co-cultures. Glia, 2005, 49:349~359
22 Kimelberg HK, Nestor NB, Feustel PJ, Inhibition of release of taurine and excitatory amino acids in ischemia and neuroprotection, Neurochem Res, 2004, 29:267~274
23 Kitagawa K, Matsumoto M, Tagaya M, et al Ischemic tolerance. phenomenon found in the brain. Brain Res, 1990, 528:21~24
24 Kosugi T, Kawahara K, Yamada T, et al Functional significance of thepreconditioninginduced down-regulation of glutamate transporter GLT-1 in neuron/astrocyte co-cultures, Neurochem Res, 2005, 30(9):1109~1116
25 Lehre KP, Levy LM, Ottersen OP, et al Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci, 1995, 15:1835~1853
26 Mehta SL, Manhas N, Raghubir R, Molecular targets in cerebral ischemia for developing novel therapeutics. Brain Research Reviews, 2007, 54:34~66
27 Mitani A, Tanaka K, Functional changes of glial glutamate transporter GLT-1 during ischemia: an in vivo study in the hippocampal CA1 of normal mice and mutant mice lacking GLT-1. J Neurosci, 2003, 23:7176~7182
28 Naoki Nakata, Hiroyuki Kato, Kyuya Kogure, Ischemic tolerance and extracellular amino acid concentrations in gerbil hippocampus measured by intracerebral microdialysis. Brain Research Bulletin, 1994, 35(3):247~251
29 Nelson RM, Lambert DG, Richard Green A, et al Pharmacology of ischemia-induced glutamate efflux from rat cerebral cortex in vitro. Brain Res, 2003, 964:1~8
30 Nicotera P, Bano D. The enemy at the gates. Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell, 2003, 115(7):768~770
31 Nishino K, Nowak TS, Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning. J Cereb Blood Flow Metab, 2004, 24(2):167~178
32 Nyitrai G, Ke′kesi KA, Juha′sz G, Extracellular level of GABA and Glu: in vivo microdialysis-HPLC measurements. Curr Top Med Chem, 2006, 6:935~940
33 O’Regan MH, Song D, VanderHeide SJ, et al Free radicals and the ischemia-evoked extracellular accumulation of amino acids in rat cerebralcortex. Neurochem. Res, 1997, 22:273~280
34 O’Regan MH, Smith-Barbour M, Perkins LM, et al A possible role for phospholipases in the release of neurotransmitter amino acids from ischemic rat cerebral cortex. Neurosci Lett, 1995, 185:191~194
35 O'Shea RD, Roles and regulation of glutamate transporters in the central nervous system. Clin Exp Pharmacol Physiol, 2002, 29(11):1018~1023
36 Phillis JW, Ren J, O’Regan MH, Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with dl-threo-beta-benzyloxyaspartate, Brain Res, 2000, 880: 224~228
37 Phillis JW, Song D, Guyot LL, et al Lactate reduced amino acid release and fuels recovery of function in the ischemic brain. Neurosci Lett, 1999, 272:195~198
38 Romera C, Hurtado O, Botella SH, et al In vitro ischemic tolerance involves upregulation of glutamate transport partly mediated by the TACE/ADAM17-tumor necrosis factor-alpha pathway. J Neurosci, 2004, 24(6):1350~1357
39 Rossi DJ, Oshima T, Attwell D, Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature, 2000, 403:316~321
40 Rothstein JD, Dykes-Hoberg M, Pardo CA, et al Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron, 1996, 16:675~686
41 Schousboe A, Sarup A, Bak LK, et al Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission. Neurochem Int, 2004, 45:521~527
42 Seki Y, Feustel PJ, Keller Jr RW, et al Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker. Stroke, 1999, 30:433~440
43 Sherwin AL, Neuroactive amino acids in focally epileptic human brain: a review. Neurochem Res, 1999, 24:1387~1395
44 Shuaib A, Ijaz MS, Miyashita H, et al GABA and glutamate levels in the substantia nigra reticulata following repetitive cerebral ischemia in gerbils.Exp Neurol, 1997, 147:311–315
45 Tanaka K, Functions of glutamate transporters in the brain. Neurosci Res, 2000, 37:15~19
46 Th¨umen A, Behnecke A, Qadri F, et al N-Methyl-norsalsolinol, a putative dopaminergic neurotoxin, passes through the blood–brain barrier in vivo. Neuroreport, 2002, 13:25~28
47 Timmerman W, Westerink BH, Brain microdialysis of GABA and glutamate: what does it signify? Synapse, 1997, 27:242~261
48 Tomimoto H, Takemoto O, Akiguchi I, et al Immunoelectron microscopic study of C-fos, C-Jun and heat shock protein after transient cerebral ischemia in gerbils. Acta Neuropathol, 1999, 97:22~30
49 Vajda FJ, Neuroprotection and neurodegenerative disease. J Clin Neurosci, 2002, 9:4~8
50 Zhou AM, Li WB, Li QJ, et al A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res, 2004, 48(4):397~404
1 Pulsinelli WA, Brierley JB. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke, 1979, 10(3): 267~272
2 陈 玲,陈海滨,吴 莹等。建立大鼠全脑缺血模型的方法与体会。汕头大学医学院学报,2000,13(1):59~60
3 范圣登,王 琛,谢 红。四血管法大鼠全脑缺血模型制作的改进。苏州大学学报(医学版)2003,23(4):416~417
4 Liu HQ, Li WB, Li QJ, et al Nitric Oxide Participates in the Induction of Brain Ischemic Tolerance via Activating ERK1/2 Signaling Pathways. Neurochem Res, 2006, 31(7): 967~974
5 Zhou AM, Li WB, Li QJ, et al A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res, 2004, 48(4): 397~404
6 Sun XC, Li WB, Li QJ, et al Limb ischemic preconditioning induces brain ischemic tolerance via p38 MAPK. Brain Res, 2006, 1084:165~174
7 Kato H, Liu Y, Araki T, et al Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res, 1991, 553:238~242
8 Grabb MC, Choi DW. Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors. J Neurosci, 1999, 19(5): 1657~1662.
9 南开大学实验动物解剖学编写组主编。实验动物解剖学。北京:人民卫生出版社,1979: 118~119
1 Pulsinelli W, Brierley J. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke, 1979, 10(3): 267~272
2 杨安峰、王平. 大鼠的解剖和组织。北京:科学出版社,1985: 118~119
3 Greene E. Anatomy of the rat. New York:Hafner Publishing Company, 1963: 253-253
1 Anderson CM, Swanson RA. Astrocyte glutamate transport: review of properties, regulation, and physiological function. Glia, 2000, 32(1):1~14
2 Aronica E, Gorter JA, Ijlst-Keizers H, et al Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins, Eur J Neurosci, 2003, 17:2106~2118
3 Atochin DN, Clark J, Demchenko IT, et al Rapid cerebral ischemic preconditioning in mice deficient in endothelial and neuronal nitric oxide synthases. Stroke, 2003, 34(5):1299~1303
4 Beart PM, O'Shea RD, Transporters for L-glutamate: An update on their molecular pharmacology and pathological involvement. British Journal of Pharmacology, 2007, 150:5~17
5 Benveniste H, Drejer J, Schousboe A, et al Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem, 1984, 43(5):1369~1374
6 Blandini F, Porter RH, Greenamyre JT. Glutamate and Pankinson`s disease. [review] Mol Neurobiol, 1996, 12:73~94
7 Boche D, Cunningham C , Gauldie J, et al Transforming growth factor-beta 1-mediated neuroprotection against excitotoxic injury in vivo. J Cereb Blood Flow Metab, 2003, 23(10):1174~1182
8 Chapman AG, Glutamate and epilepsy. J Nutr, 2000, 130:1043~1045
9 Carlson M, Carlson A. Interactions between glutamatergic and monoaminergic systems within the basal ganglia—implications for schizophrenia and Parkinson’s disease. Trends Neurosci, 1990, 13:272~276
10 Chaudhry FA, Lehre KP, Lookeren Campagne MV, et al Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocyto- chemistry. Neuron, 1995, 15:711~720
11 Chen W, Mahadomrongkul V, Berger UV, et al Aoki C and Rosenberg PA, The glutamate transporter GLT1a is expressed in excitatory axon terminals of mature hippocampal neurons. J Neurosci, 2004, 24: 1136~1148
12 Chen W, Aoki C, Mahadomrongkul V, et al Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain. J Neurosci, 2002, 22(6):2142~2152
13 Dirnagl U, Simon RP, Hallenbeck JM. Ischemic tolerance and endogenous neuroprotection. Trends Neurosci, 2003, 26(5):248~254
14 Danbolt, N.C. Glutamate uptake. Prog Neurobiol, 2001, 65:1~105
15 Davis KE, Straff DJ, Weinstein EA, et al Multiple signaling pathways regulate cell surface expression and activity of the excitatory amino acid carrier 1 subtype of Glu transporter in C6 glioma. J Neurosci, 1998, 18:2475~2485
16 Douen AG, Akiyama K, Hogan MJ, et al Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebral cortex plasma membranes. J Neurochem, 2000, 75(2):812~818
17 During MJ, Spencer DD. Extracellular lippocampal glutamate and spontaneous seizure in the conscious human brain. Lancet, 1993, 341:607~1610
18 Euler T, Wassle H. Immunocytochemical identification of cone bipolarcells in the rat retina. J Comp Neurol, 1995, 361:461~478
19 Feustel PJ, Jin Y, Kimelberg HK, Volume-regulated anion channels are the predominant contributors to release of excitatory amino acids in the ischemic cortical penumbra. Stroke, 2004, 35(5):1164~1168
20 Figiel M, Engele J. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a Neuron-Derived Peptide Regulating Glial Glutamate Transport and Metabolism. J Neurosci, 2000, 20(10):3596~3605
21 Figiel M, Maucher T, Rozyczka J, et al Regulation of glial glutamate transporter expression by growth factors. Exp Neurol, 2003, 183:124~135
22 Futura A, Rothstein JD, Martin LJ, Glutamate Transporter Protein Subtypes Are Expressed Differentially during Rat CNS Development. J Neurosci, 1997, 17:8363~8375
23 Garcia L, Burda J, Hrehorovska M, et al Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression. J Neurochem, 2004, 88(1):136~147
24 Gegelashvili G, Civenni G, Racagni G, et al Glutamate receptor agonists up-regulate the glutamate transporter GLAST in astrocytes. Neuroreport, 1996, 8:261~265
25 Gegelashvili G, Danbolt N C, Schousboe A. Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. J Neurochem, 1997, 69:2612~2615
26 Gegelashvili G, Robinson MB, Trotti D, et al Regulation of glutamate transporters in health and disease, Prog Brain Res, 2001, 132:267~286
27 Gegelashvili G, Schousboe A Cellular distribution and kinetic properties of high-affinity glutamate transporters. Brain Res Bull, 1998, 45:233~238
28 Gogas KR, Glutamate-based therapeutic approaches: NR2B receptor antagonists. Curr Opin Pharmacol, 2006, 6(1):68~74.
29 Grabb MC, Lobner D, Turetsky DM, et al Preconditioned resistance to oxygen-glucose deprivation-induced cortical neuronal death: alterations in vesicular GABA and glutamate release. Neuroscience, 2002,115(1):173~183
30 Greenamyre JT, Young AB. Excitatory amino acids and Alzheimer’s disease. Neurobiol. Aging, 1989, 10:593~602
31 Guo H, Lai L, Butchbach ME, et al Increased expression of the glial glutamate transporter EAAT2 modulates excitotoxicity and delays the onset but not the outcome of ALS in mice. Hum Mol Genet, 2003, 12(19):2519~2532
32 Hayashi M, Hayashi R, Tanii H, et al The influence of neuronal cells on the development of glutamine synthetase in astrocytes in vitro. Dev Brain Res, 1988, 41:37~42
33 Heurteaux C, Lauritzen I, Widmann C, et al Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning. PNAS, 1995, 92:4666~4670
34 Hong JS, McGinty JF, Grimes L, et al Seizure-induced alterations in the metabolism of hippocampal opioid peptides suggest opioid modulation of seizure-related behaviors. NIDA Res Monogr, 1988, 82:48~66
35 Hu WH, Walters WM, Xia XM, et al Neuronal glutamate transporter EAAT4 is expressed in astrocytes. Glia, 2003, 44(1):13~25
36 Kalandadze A, Wu Y, Robinson MB. Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486. J Biol Chem, 2002, 29;277(48):45741~45750.
37 Kanai Y, Hediger MA. The glutamate and neutral amino acid transporter family: physiological and pharmacological implications. Eur J Pharmacol, 2003, 479(1-3):237~247
38 Kanai Y, Hediger MA. The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects. Pflugers Arch, 2004, 447(5):469~479
39 Kanai Y, Trotti D, Nussberger S, et al The high affinity glutamate transporter family. In: Neurotransmitter transporters: structure, function, and regulation (Reith MEA, ed), 1997, pp171–213. Totowa, NJ: Humana.
40 Kato H, Kogure K. Biochemical and molecular characteristics of the brain with developing cerebral infarction. Cell Mol Neurobiol, 1999, 19:93~108
41 Katsuki H, Akaike A. Excitotoxic degeneration of hypothalamic orexin neurons in slice culture. Neurobiol Dis, 2004, 15(1):61~69
42 Kosugi T, Kawahara K, Reversed actrocytic GLT-1 during ischemia is crucial to excitotoxic death of neurons, but contributes to the survival of astrocytes themselves, Neurochem Res, 2006, 31(7):933~943
43 Lee J, Lee J E, Cho EH, et al An essential histidine residue in GTP binding domain of bovine brain glutamate dehydrogenase isoproteins. Mol Cells, 2001, 12:121~126
44 Lehre KP, Danbolt NCThe number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci, 1998, 18:8751~8757
45 Lehre K P, Levy LM, Ottersen OP, et al Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci, 1995, 15:1835~1853
46 Levenson J, Endo S, Kategaya LS, et al Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia. Proc Natl Acad Sci USA, 2000, 97:12858~12863
47 Levenson J, Weeber E, Selcher JC, et al Long-term potentiation and contextual fear conditioning increase neuronal glutamate uptake. Nat Neurosci, 2002, 5:155~161
48 Lin CL, Bristol LA, Jin L, et al Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron, 1998, 20(3):589~602
49 Lipton P. Ischemic cell death in brain neurons. Physiol Rev, 1999, 79:1431~1568
50 Liu GJ, Madsen BW. PACAP-38 modulates activity of NMDA receptors in cultured chick cortical neurons. J Neurophysiol, 1997, 78:2231~2234
51 Maleszka R, Helliwell P, Kucharski R. Pharmacological interference with glutamate re-uptake impairs long-term memory in the honeybee, apis mellifera. Behav Brain Res, 2000, 115:49~53
52 Mansouri FA, Motamedi F, Fathollahi Y. Chronic in vivo morphine administration facilitates primed-bursts-induced long-term potentiation of Schaffer collateral-CA1 synapses in hippocampal slices in vitro. Brain Res, 1999, 815:419~423
53 Maragakis NJ, Dykes-Hoberg M, Rothstein JD. Altered expression of the glutamate transporter EAAT2b in neurological disease. Ann Neurol, 2004, 55:469~477
54 Martin J-L, Gasser D, Magistretti PJ. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide potentiate c-fos expression induced by glutamate in cultured cortical neurons. J Neurochem, 1995, 65:1~9.
55 Masliah E, Alford M, DeFeresa R, et al Deficient glutamate transport is associated with neurodegeneration in Alzheimer`s disease. Ann Neurol 1996, 40:759~766
56 Masliah E, Alford M, Mallory M, et al Abnormal glutamate transport function in mutant amyloid precursor protein transgenic mice. Exp Neurol, 2000, 163(2):381~387
57 Mathern GW, Menoza D, Lozada A, et al Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy. Neurology, 1999, 52:453~472
58 Matsumoto Y, Yamamoto S, Suzuki Y, et al. Na+/H+ exchanger inhibitor, SM-20220, is protective against excitotoxicity in cultured cortical neurons. Stroke, 2004, 35(1):185~190
59 Meldrum BS. The role of glutamate in epilepsy and other disorders. [Review] Neurology 1994, 44:S14~23
60 Meldrum BS, Akbar MT, Chapman AG. Glutamate receptors and transporters in genetic and acquired models of epilepsy. [Review] Epilepsy Res, 1999, 36:189~204
61 Mitani A, Tanaka K. Functional Changes of Glial Glutamate Transporter GLT-1 during Ischemia: An In Vivo Study in the Hippocampal CA1 of Normal Mice and Mutant Mice Lacking GLT-1. J Neurosci, 2003, 23(18):7176~7182
62 Munch C, Ebstein M, Seefried U, et al Alternative splicing of the 5'-sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis. J Neurochem, 2002, 82(3):594~603
63 Namura S, Maeno H, Takami S, et al Inhibition of glial glutamate transporter GLT-1 augments brain edema after transient focal cerebral ischemia in mice. Neurosci Lett, 2002, 324(2):117~120
64 Nedergaard M, Takano Y, Hansen AJ. Beyond the role of glutamate as a neurotransmitter. Nat Rev Neurosci, 2002,3(9):748~755
65 Neugebauer V, Glutamate receptor ligands. Handb Exp Pharmacol, 2007, 177:217~249
66 Nicotera P, Bano D. The enemy at the gates. Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell, 2003, 115(7):768~770
67 Nishino K, Nowak Jr TS. Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning. J Cereb Blood Flow Metab. 2004, 24(2):167~178
68 Ng KT, O'Dowd BS, Rickard NS, et al Complex roles of glutamate in the Gibbs-Ng model of one-trial aversive learning in the new-born chick. Neurosci Biobehav Rev, 1997, 21:45~54
69 Olney JW. Excitotoxic amino acids and neuropsychiatric disorders. Annu Rev Pharmacol Toxicol, 1990, 30:47~71
70 O'Shea RD. Roles and regulation of glutamate transporters in the central nervous system. Clin Exp Pharmacol Physiol, 2002, 29(11):1018~1023
71 Payet O, Maurin L, Bonne C, et al Hypoxia stimulates glutamate uptake in whole rat retinal cells in vitro. Neurosci Lett, 2004, 356(2):148~150
72 Pellegri G, Magistretti PJ, Martin J-L. VIP and PACAP potentiate the action of glutamate on BDNF expression in mouse cortical neurones. Eur J Neurosci, 1998, 10:272~280.
73 Perego C, Vanoni C, Bossi M, et al The GLT-1 and GLAST Glutamate Transporters Are Expressed on Morphologically Distinct Astrocytes and Regulated by Neuronal Activity in Primary Hippocampal Cocultures. J Neurochem, 2000, 75(3):1076~1084
74 Perry TL, Hansen S. What excitotoxin kills striatal neurons in Huntington’s disease? Clues from neurochemical studies. Neurology, 1990, 40:20~24
75 Proper EA, Hoogland G, Kappen SM, et al Distribution of glutamate transporters in the hippocampus of patients with pharmaco-resistant temporal lobe epilepsy. Brain, 2002,125:32~43
76 Pu L, Bao GB, Xu NJ, et al Hippocampal long-term potentiation is reduced by chronic morphine treatment and can be restored by re-exposure to opiates. J Neurosci, 2002, 22:1914~1921
77 Rao VL, Dogan A, Todd KG, et al Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain. J Neurosci, 2001a, 21(6):1876~1783
78 Rao VL, Dogan A, Bowen KK, et al Antisense knockdown of the glial glutamate transporter GLT-1 exacerbates hippocampal neuronal damage following traumatic injury to rat brain. Eur J Neurosci, 2001b, 13(1):119~128
79 Rauen T, Kanner BI. Localization of the glutamate transporter GLT-1 in rat and macaque monkey retinae. Neurosci Lett, 1994, 169:137~140
80 Rauen T. Diversity of glutamate transporter expression and function in the mammalian retina. Amino Acids, 2000, 19:53~62
81 Raval AP, Dave KR, Mochly-Rosen D, et al Epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice. J Neurosci, 2003,23(2):384~391
82 Reye P, Sullivan R, Fletcher EL, et al Distribution of two splice variants of the glutamate transporter GLT1 in the retinas of humans, monkeys, rabbits, rats, cats, and chickens. J Comp Neurol, 2002a, 445:1~12
83 Reye P, Sullivan R, Pow DV. Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina. J Comp Neurol, 2002b, 447:323~330
84 Rintoul GL, Filiano AJ, Brocard JB, et al Glutamate decreases mitochondrial size and movement in primary forebrain neurons. J Neurosci, 2003, 23(21):7881~7888
85 Robinson MB, Dowd LA. Heterogeneity and functional properties of subtypes of sodium-dependent glutamate transporters in the mammalian central nervous system. Adv Pharmacol, 1997, 37:69~115
86 Romera C, Hurtado O, Botella SH, et al In vitro ischemic tolerance involves upregulation of glutamate transport partly mediated by the TACE/ADAM17-tumor necrosis factor-alpha pathway. J Neurosci, 2004, 24(6):1350~1357
87 Rothstein JD, Dykes-Hoberg M, Pardo CA, et al Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron, 1996, 16:675~686
88 Rothstein JD, Patel S, Regan MR, et al -Lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature, 2005, 433:73~77
89 Rothstein JD, Van Kammen M, Levy AI, et al Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann Neurol, 1995, 38:73~84
90 Rozyczka J, Figiel M, Engele J. Endothelins negatively regulate glial glutamate transporter expression. Brain Pathol, 2004, 14(4):406~414
91 Sanchez-Gomez MV, Alberdi E, Ibarretxe G, et al Caspase-dependent and caspase-independent oligodendrocyte death mediated by AMPA and kainate receptors. Neurosci, 2003, 23(29):9519~9528
92 Schlag BD, Vondrasek JR, Munir M, et al Regulation of glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons. Mol Pharmacol, 1998, 53:355~369
93 Shigery Y, Seal RP, Shimamoto K. Molecular pharmacology of glutamate transporters, EAATs and VGLUTs. Brain Res Brain Res Rev, 2004, 45(3):250~265
94 Shobha K, Vijayalakshmi K, Alladi PA, et al Altered in-vitro and in-vivo expression of glial glutamate transporter-1 following exposure to cerebrospinal fluid of amyotrophic lateral sclerosis patients. J Neurol Sci, 2007, 254(1-2):9~16.
95 Sonnewald U, Westergaard N, Schousboe A. Glutamate transport and metabolism in astrocytes. Glia, 1997, 21:56~63
96 Stella N, Magistretti PJ. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) potentiate the glutamate-evoked release of arachidonic acid from mouse cortical neurons. Evidence for a cAMP-independent mechanism. J Biol Chem, 1996, 271:23705~23710
97 Stoffel W, Korner R, Wachtmann D, et al Functional analysis of glutamate transporters in excitatory synaptic transmission of GLAST1 and GLAST1/EAAC1 deficient mice. Mol Brain Res, 2004, 128:170~181
98 Suchak SK, Baloyianni NV, Perkinton MS, et al The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings. J Neurochem, 2003, 84(3):522~32
99 Sullivan R, Rauen T, Fischer F, et al Cloning, transport properties, and differential localization of two splice variants of GLT-1 in the rat CNS: Implications for CNS glutamate homeostasis. Glia, 2004, 45(2):155~169
100 Sun Y, Jin K, Peel A, et al Greenberg DA. Neuroglobin protects the brain from experimental stroke in vivo. PNAS, 2003; 100(6):3497~3500
101 Swanson RA, Liu J, Miller JW, et al Neuronal regulation of glutamate transporter subtype expression in astrocytes. J Neurosci, 1997, 17:932~940
102 Szatkowski M, Attwell D. Triggering and execution of neuronal death in brain ischemia: Two phases of glutamate release by different mechanisms. Trends Neurosci, 1994, 17:359~365
103 Takagi K, Ginsberg MD, Globus MY, et al Effect of hyperthermia on glutamate release in ischemic penumbra after middle cerebral artery occlusion in rats. Am J Physiol, 1994, 267:H1770~1776
104 Tanaka K, Watase K, Manabe T, et al Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science, 1997, 276:1699~1702
105 Tanaka K. Functions of glutamate transporters in the brain. Neurosci Res, 2000, 37:15~19
106 Tauskela JS, Brunette E, Monette R, et al Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling. Am J Physiol Cell Physiol, 2003, 285(4):C899~911
107 Tessler S, Danbolt NC, Faull RL, et al Expression of the glutamate transporters in human temporal lobe spilepsy. Neurossience, 1999, 88:1083~1091
108 Thorlin T, Roginski RS, Choudhury K, et al Regulation of the glial glutamate transporter GLT-1 by glutamate and delta-opioid receptor stimulation. FEBS Lett, 1998, 425:453~459
109 Uchiyama-Tsuyuki Y, Araki H, Yae T, et al Changes in the extracellular concentrations of amino acids in the rat striatum during transient focal cerebral ischemia. J Neurochem, 1994, 62:1074~1078
110 Ullensvang K, Lehre K P, Storm-Mathisen J, et al Differential developmental expression of the two rat brain glutamate transporter proteins GLAST and GLT. Eur J Neurosci, 1997, 9:1646~1655
111 Van Damme P, Dewil M, Robberecht W, et al Excitotoxicity and amyotrophic lateral sclerosis. Neurodegener Dis, 2005, 2(3-4):147~159
112 Van den Bosch L, The causes and mechanism of selective motor neuron death in amyotrophic lateral sclerosis. Werh K Acad Geneeskd Belg,2006, 68(4):249~269
113 Velly LJ, Guillet BA, Masmejean FM, et al Neuroprotective effects of propofol in a model of ischemic cortical cell cultures: role of glutamate and its transporters. Anesthesiology, 2003, 99(2):368~375
114 Watase K, Hashimoto K, Kano M, et al Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. Eur J Neurosci, 1998, 10:976~988
115 Whetsell WO Jr J. Current concepts of excitotoxicity. Neuropathol Exp Neurol, 1996, 55(1):1~13
116 Whetsell Jr WO, Shapira NA. Neuroexcitation, excitotoxicity, and human neurological disease. Lab Invest, 1993, 68:372~387
117 Williams JT, Christie MJ, Manzoni O. Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev, 2001, 81:99~343
118 Wilson CL, Maidment NT, Shomer MH, et al Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy. Epilepsy Res, 1996, 265:245~254
119 Won MH, Kang T-C, Jeon GS, et al Immunohistochemical detection of oxidative DNA damage induced by ischemia-reperfusion insults in gerbil hippocampus in vivo. Brain Res, 1999, 836:70~78
120 Won MH, Kang TC, Park SK, et al The alterations of N-Methyl-D-aspartate receptor expressions and oxidative DNA damage in the CA1 area at the early time after ischemia-reperfusion insult. Neurosci Lett, 2001, 301:139~142
121 Xu NJ, Bao L, Fan HP, et al Morphine Withdrawal Increases Glutamate Uptake and Surface Expression of Glutamate Transporter GLT1 at Hippocampal Synapses. J Neurosci, 2003, 23(11):4775~4784
122 Yamada K, Watanabe M, Shibata T, et al Glutamate transporter GLT-1 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression. J Neurosci, 1998, 18:5706~5713
123 Young KC, McGehee DS, Brorson JR. Glutamate receptor expression andchronic glutamate toxicity in rat motor cortex. 2007, Epub ahead of print
124 Zacco A, Togo J, Spence K, et al 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitors protect cortical neurons from excitotoxicity. Neuroscience, 2003, 23(35):11104~11111
125 Zelenaia O, Schlag BD, Gochenauer GE, et al Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB. Mol Pharmacol, 2000, 57:667~678
126 Zhou AM, Li WB, Li QJ, et al A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res, 2004, 48(4):379~404
2 Atochin DN, Clark J, Demchenko IT, et al Rapid cerebral ischemic preconditioning in mice deficient in endothelial and neuronal nitric oxide synthases. Stroke, 2003, 34(5):1299~1303
3 Chen J, Graham SH, Zhu RL, et al Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab, 1996, 16:566~577
4 Chen JC, Hcu-Chou H, Lu JL, et al Down-regulation of the glial glutamate transporter GLT-1 in rat hippocampus and striatum and its modulation by a group III metabotropic glutamate receptor antagonist following transient global forebrain ischemia. Neuropharmacology, 2005, 49(5):703~714
5 Fairman WA, Vandenberg RJ, Arriza JL, et al An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature, 1995, 375:599~603
6 Garcia L, Burda J, Hrehorovska M, et al Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression. J Neurochem, 2004, 88(1):136~147
7 Heurteaux C, Lauritzen I, Widmann C, et al Essential role of adenosine,adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning. PNAS, 1995, 92: 4666~4670
8 Kato H, Liu Y, Araki T, et al Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res, 1991, 553:238~242
9 Kato H, Kogure K, Biochemical and molecular characteristics of the brain with developing cerebral infarction. Cell Mol Neurobiol, 1999, 19:93~108
10 Kawahara K, Kosugi T, Tanaka M, et al Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning- induced ischemic tolerance of neurons in neuron/astrocyte co-cultures. Glia, 2005, 49:349~359
11 Kirino T, Ischemic tolerance. J Cereb Blood Flow Metab, 2002, 22:1283~2196
12 Kitagawa K, Matsumoto M, Tagaya M, et al ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res, 1990, 528:21~24
13 Kosugi T, Kawahara K, Yamada T,et al Functional significance of the preconditioning-induced down-regulation of glutamate transporter GLT-1 in neuron/astrocyte co-cultures. Neurochem res, 2005, 30(9):1109~1116
14 Lehre KP, Levy LM, Ottersen OP, et al Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocyto- chemical observations. J Neurosci, 1995, 3:1835~1853
15 Levy LM, Lehre KP, Rolstad B, et al A monoclonal antibody raised against an [Na+ -K+] coupled L-glutamate transporter purified from rat brain confirms glial cell localization. FEBS Lett, 1993, 317:79~84
16 Lipton P, Ischemic cell death in brain neurons. Physiol Rev, 1999, 79:1431~1568
17 Liu HQ, Li WB, Li SQ, et al Nitric oxide participates in the induction of brain ischemic tolerance via activating ERK1/2 signaling pathways. Neurochem Res, 2006, 31(7):967~974
18 Mitani A, Tanaka K. Functional Changes of Glial Glutamate transporterGLT-1 during Ischemia: An In Vivo Study in the Hippocampal CA1 of Normal Mice and Mutant Mice Lacking GLT-1. J Neurosci, 2003, 23(18):7176~7182
19 Neary JT, McCarthy M, Kang Y, et al Mitogenic signaling from P1 and P2 purinergic receptors to mitogen-activated protein kinase in human fetal astrocytes. Neurosci Letters, 1998, 242:159~162
20 Neary JT, Kang Y, Shi YF, et al P2 receptor signalling, proliferation of astrocytes, and expression of molecules involved in cell-cell interactions. Novartis Found Symp, 2006, 276:131-43; discussion 143-7, 233-7, 275-81
21 Nicotera P, Bano D. The enemy at the gates: Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell, 2003, 115(7):768~770
22 Nishino K, Nowak Jr TS. Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning. J Cereb Blood Flow Metab, 2004, 24(2):167~178
23 Payet O, Maurin L, Bonne C, et al Hypoxia stimulates glutamate uptake in whole rat retinal cells in vitro. Neurosci Lett, 2004, 356(2):148~150
24 Pines G, Danbolt NC, Bj?r?s M, et al Cloning and expression of a rat brain L-glutamate transporter. Nature, 1992, 360:464~467
25 Pulsinelli WA, Brierley JB. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke, 1979, 10(3):267~272
26 Raghavendra Rao VL, Rao AM, Dogen A, et al Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia. Neurochem Int, 2000, 36(6):531~537
27 Rao L, Dogan A, Todd KG, et al Antisense Knockdown of the Glial Glutamate Transporter GLT-1, But Not the Neuronal Glutamate Transporter EAAC1, Exacerbates Transient Focal Cerebral Ischemia-Induced Neuronal Damage in Rat Brain. J Neurosci, 2001, 21(6):1876~1883
28 Rao VL, Dogan A, Todd KG, et al Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain. J Neurosci, 2001a, 21(6):1876~1883
29 Rao VL, Dogan A, Bowen KK, et al Antisense knockdown of the glial glutamate transporter GLT-1 exacerbates hippocampal neuronal damage following traumatic injury to rat brain. Eur J Neurosci, 2001b, 13(1):119~128
30 Rauen T, Kanner BI. Localization of the glutamate transporter GLT-1 in rat and macaque monkey retinae. Neurosci Lett, 1994, 169:137~140
31 Romera C, Hurtado O, Botella SH, et al In vitro ischemic tolerance involves upregulation of glutamate transport partly mediated by the TACE/ADAM17-tumor necrosis factor-alpha pathway. J Neurosci, 2004,
24(6):1350~1357
32 Rossi DJ, Oshima T, Attwell D. Glutamate release in severe brain ischaemia is mainly by reversed uptake, Nature, 2000, 403:316~321
33 Rothstein JD, Martin L, Levey AI, et al Localization of neuronal and glial glutamate transporters. Neuron, 1994, 13:713~725
34 Rothstein JD, Dykes-Hoberg M, Pardo CA, et al Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron, 1996, 16:675~686
35 Seki Y, Feustel PJ, Keller RW Jr, et al Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker. Stroke, 1999, 30(2):433~440
36 Storck T, Schulte S, Hofmann K, et al Structure, expression, and functional analysis of a Na+-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci USA, 1992, 89:10955~10959
37 Tanaka K, Watase K, Manabe T, et al Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science, 1997, 276:1699~1702
38 Tomimoto H, Takemoto O, Akiguchi I, et al Immunoelectron microscopicstudy of C-fos, C-Jun and heat shock protein after transient cerebral ischemia in gerbils. Acta Neuropathol 1999, 97:22~30
39 Yamada K, Watanabe M, Shibata T, et al Glutamate transporter GLT-1 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression. J Neurosci, 1998, 18:5706~5713
40 Yeh TH, Hwang HM, Chee JJ, et al Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia. Neurobiol Dis, 2005, 18(3):476~483
41 Zacco A, Togo J, Spence K, et al 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitors protect cortical neurons from excitotoxicity. Neuroscience, 2003, 23(35):11104~11111
42 Zhou AM, Li WB, Li QJ, et al A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res, 2004, 48(4): 379~404
1 Benveniste H, Drejer J, Schousboe A, et al Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem, 1984, 43(5):1369~1374
2 Hossmann, KA, Thresholds of ischemic injury. In: Ginsberg, MD, Bogousslavsky, J (Eds.), Cerebrovascular Disease. Pathophysiology, Diagnosis, and Treatment, 1998, vol. 1. Blackwell Science, Malden, MA, USA, pp. 193~204
3 Liu S, Lau L, Wei J, et al Expression of Ca2+-permeable AMPA receptor channels primes cell death in transient forebrain ischemia. Neuron, 2004, 43: 43~55
4 Nicotera P, Bano D, The enemy at the gates: Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell, 2003, 115(7):768~770
5 Schmidt-Kastner R, Freund TF, Selective vulnerability of the hippocampus in brain ischemia. Neuroscience, 1991, 40: 599~636
6 Yeh TH, Hwang HM, Chee JJ, et al Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia. Neurobiol Dis, 2005, 18(3):476~483
1 Atochin DN, Clark J, Demchenko IT et al Rapid cerebral ischemicpreconditioning in mice deficient in endothelial and neuronal nitric oxide synthases. Stroke, 2003, 34(5):1299~1303
2 Attwell D, Barbour B, Szatkowski M, Nonvesicular release of neurotransmitter. Neuron, 1993, 11:401~407
3 Baker DA, Xi ZX, Shen H, et al The origin and neuronal function of in vivo nonsynaptic glutamate. J Neurosci, 2002, 22: 9134~9141
4 Benveniste H, The excitotoxin hypothesis in relation to cerebral ischemia, Cerebrovasc. Brain Metab Rev, 1991, 3:213~245
5 Bradford HF, Glutamate, GABA and epilepsy. Prog. Neurobiol, 1995, 47: 477~511
6 Bradford SE, Nadler JV, Aspartate release from rat hippocampal synaptosomes. Neuroscience, 2004, 128(4):751~65
7 Chatterton JE, Awobuluyi M, Premkumar LS, et al Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits. Nature, 2002, 415:793~798.
8 Chen J, Graham SH, Zhu RL, et al Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab, 1996, 16:566~77
9 Choi DW, Rothman SM, The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Rev Neurosci, 1990, 13:171~182
10 French ED, Vezzani A, Whetsell Jr WO, et al Antiexcitotoxic actions of taurine in the rat hippocampus studied in vivo and in vitro. Adv Exp Med Biol, 1986, 203:349~362
11 Garcia L, Burda J, Hrehorovska M, et al Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression. J Neurochem, 2004, 88(1):136~47
12 Gemba T, Matsunaga K, Ueda M, Changes in extracellular concentration of amino acids in the hippocampus during cerebral ischemia in stroke-prone SHR, stroke-resistant SHR and normotensive rats. Neurosci Lett, 1992, 135: 184~188
13 Hertz L, Zielke HR, Astrocytic control of glutamatergic activity:astrocytes as stars of the show. Trends Neurosci, 2004, 27:735~743
14 Kirino T, Ischemic tolerance. J Cereb Blood Flow Metab, 2002, 22:1283~1296
15 Hillered L, Hallstrom A, Segersvard S, et al Dynamics of extracellular metabolites in the striatum after middle cerebral artery occlusion in the rat monitored by intracerebral microdialysis. J Cereb Blood Flow Metab, 1989, 9:607~616
16 Huang YH, Bergles DE, Glutamate transporters bring competition to the synapse. Curr Opin Neurobiol, 2004, 14:346~352
17 Johansen EF, Diemer NH, Enhancement of GABA neurotransmission after cerebral ischemia in the rat reduces loss of hippocampal CA1 pyramidal cells. Acta Neurol Scand, 1991, 84:1~6
18 Katsuki H, Akaike A. Excitotoxic degeneration of hypothalamic orexin neurons in slice culture. Neurobiol Dis, 2004, 15(1):61~9
19 Liu HQ, Li WB, Li SQ, et al Nitric oxide participates in the induction of brain ischemic tolerance via activating ERK1/2 signaling pathways. Neurochem Res, 2006, 31(7):967~974
20 Kato H, Liu Y, Araki T, et al Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage followingsecondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res, 1991, 553:238~242
21 Kawahara K, Kosugi T, Tanaka M, et al Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning- induced ischemic tolerance of neurons in neuron/ astrocyte co-cultures. Glia, 2005, 49:349~359
22 Kimelberg HK, Nestor NB, Feustel PJ, Inhibition of release of taurine and excitatory amino acids in ischemia and neuroprotection, Neurochem Res, 2004, 29:267~274
23 Kitagawa K, Matsumoto M, Tagaya M, et al Ischemic tolerance. phenomenon found in the brain. Brain Res, 1990, 528:21~24
24 Kosugi T, Kawahara K, Yamada T, et al Functional significance of thepreconditioninginduced down-regulation of glutamate transporter GLT-1 in neuron/astrocyte co-cultures, Neurochem Res, 2005, 30(9):1109~1116
25 Lehre KP, Levy LM, Ottersen OP, et al Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci, 1995, 15:1835~1853
26 Mehta SL, Manhas N, Raghubir R, Molecular targets in cerebral ischemia for developing novel therapeutics. Brain Research Reviews, 2007, 54:34~66
27 Mitani A, Tanaka K, Functional changes of glial glutamate transporter GLT-1 during ischemia: an in vivo study in the hippocampal CA1 of normal mice and mutant mice lacking GLT-1. J Neurosci, 2003, 23:7176~7182
28 Naoki Nakata, Hiroyuki Kato, Kyuya Kogure, Ischemic tolerance and extracellular amino acid concentrations in gerbil hippocampus measured by intracerebral microdialysis. Brain Research Bulletin, 1994, 35(3):247~251
29 Nelson RM, Lambert DG, Richard Green A, et al Pharmacology of ischemia-induced glutamate efflux from rat cerebral cortex in vitro. Brain Res, 2003, 964:1~8
30 Nicotera P, Bano D. The enemy at the gates. Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell, 2003, 115(7):768~770
31 Nishino K, Nowak TS, Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning. J Cereb Blood Flow Metab, 2004, 24(2):167~178
32 Nyitrai G, Ke′kesi KA, Juha′sz G, Extracellular level of GABA and Glu: in vivo microdialysis-HPLC measurements. Curr Top Med Chem, 2006, 6:935~940
33 O’Regan MH, Song D, VanderHeide SJ, et al Free radicals and the ischemia-evoked extracellular accumulation of amino acids in rat cerebralcortex. Neurochem. Res, 1997, 22:273~280
34 O’Regan MH, Smith-Barbour M, Perkins LM, et al A possible role for phospholipases in the release of neurotransmitter amino acids from ischemic rat cerebral cortex. Neurosci Lett, 1995, 185:191~194
35 O'Shea RD, Roles and regulation of glutamate transporters in the central nervous system. Clin Exp Pharmacol Physiol, 2002, 29(11):1018~1023
36 Phillis JW, Ren J, O’Regan MH, Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with dl-threo-beta-benzyloxyaspartate, Brain Res, 2000, 880: 224~228
37 Phillis JW, Song D, Guyot LL, et al Lactate reduced amino acid release and fuels recovery of function in the ischemic brain. Neurosci Lett, 1999, 272:195~198
38 Romera C, Hurtado O, Botella SH, et al In vitro ischemic tolerance involves upregulation of glutamate transport partly mediated by the TACE/ADAM17-tumor necrosis factor-alpha pathway. J Neurosci, 2004, 24(6):1350~1357
39 Rossi DJ, Oshima T, Attwell D, Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature, 2000, 403:316~321
40 Rothstein JD, Dykes-Hoberg M, Pardo CA, et al Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron, 1996, 16:675~686
41 Schousboe A, Sarup A, Bak LK, et al Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission. Neurochem Int, 2004, 45:521~527
42 Seki Y, Feustel PJ, Keller Jr RW, et al Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker. Stroke, 1999, 30:433~440
43 Sherwin AL, Neuroactive amino acids in focally epileptic human brain: a review. Neurochem Res, 1999, 24:1387~1395
44 Shuaib A, Ijaz MS, Miyashita H, et al GABA and glutamate levels in the substantia nigra reticulata following repetitive cerebral ischemia in gerbils.Exp Neurol, 1997, 147:311–315
45 Tanaka K, Functions of glutamate transporters in the brain. Neurosci Res, 2000, 37:15~19
46 Th¨umen A, Behnecke A, Qadri F, et al N-Methyl-norsalsolinol, a putative dopaminergic neurotoxin, passes through the blood–brain barrier in vivo. Neuroreport, 2002, 13:25~28
47 Timmerman W, Westerink BH, Brain microdialysis of GABA and glutamate: what does it signify? Synapse, 1997, 27:242~261
48 Tomimoto H, Takemoto O, Akiguchi I, et al Immunoelectron microscopic study of C-fos, C-Jun and heat shock protein after transient cerebral ischemia in gerbils. Acta Neuropathol, 1999, 97:22~30
49 Vajda FJ, Neuroprotection and neurodegenerative disease. J Clin Neurosci, 2002, 9:4~8
50 Zhou AM, Li WB, Li QJ, et al A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res, 2004, 48(4):397~404
1 Pulsinelli WA, Brierley JB. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke, 1979, 10(3): 267~272
2 陈 玲,陈海滨,吴 莹等。建立大鼠全脑缺血模型的方法与体会。汕头大学医学院学报,2000,13(1):59~60
3 范圣登,王 琛,谢 红。四血管法大鼠全脑缺血模型制作的改进。苏州大学学报(医学版)2003,23(4):416~417
4 Liu HQ, Li WB, Li QJ, et al Nitric Oxide Participates in the Induction of Brain Ischemic Tolerance via Activating ERK1/2 Signaling Pathways. Neurochem Res, 2006, 31(7): 967~974
5 Zhou AM, Li WB, Li QJ, et al A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res, 2004, 48(4): 397~404
6 Sun XC, Li WB, Li QJ, et al Limb ischemic preconditioning induces brain ischemic tolerance via p38 MAPK. Brain Res, 2006, 1084:165~174
7 Kato H, Liu Y, Araki T, et al Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res, 1991, 553:238~242
8 Grabb MC, Choi DW. Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors. J Neurosci, 1999, 19(5): 1657~1662.
9 南开大学实验动物解剖学编写组主编。实验动物解剖学。北京:人民卫生出版社,1979: 118~119
1 Pulsinelli W, Brierley J. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke, 1979, 10(3): 267~272
2 杨安峰、王平. 大鼠的解剖和组织。北京:科学出版社,1985: 118~119
3 Greene E. Anatomy of the rat. New York:Hafner Publishing Company, 1963: 253-253
1 Anderson CM, Swanson RA. Astrocyte glutamate transport: review of properties, regulation, and physiological function. Glia, 2000, 32(1):1~14
2 Aronica E, Gorter JA, Ijlst-Keizers H, et al Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins, Eur J Neurosci, 2003, 17:2106~2118
3 Atochin DN, Clark J, Demchenko IT, et al Rapid cerebral ischemic preconditioning in mice deficient in endothelial and neuronal nitric oxide synthases. Stroke, 2003, 34(5):1299~1303
4 Beart PM, O'Shea RD, Transporters for L-glutamate: An update on their molecular pharmacology and pathological involvement. British Journal of Pharmacology, 2007, 150:5~17
5 Benveniste H, Drejer J, Schousboe A, et al Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem, 1984, 43(5):1369~1374
6 Blandini F, Porter RH, Greenamyre JT. Glutamate and Pankinson`s disease. [review] Mol Neurobiol, 1996, 12:73~94
7 Boche D, Cunningham C , Gauldie J, et al Transforming growth factor-beta 1-mediated neuroprotection against excitotoxic injury in vivo. J Cereb Blood Flow Metab, 2003, 23(10):1174~1182
8 Chapman AG, Glutamate and epilepsy. J Nutr, 2000, 130:1043~1045
9 Carlson M, Carlson A. Interactions between glutamatergic and monoaminergic systems within the basal ganglia—implications for schizophrenia and Parkinson’s disease. Trends Neurosci, 1990, 13:272~276
10 Chaudhry FA, Lehre KP, Lookeren Campagne MV, et al Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocyto- chemistry. Neuron, 1995, 15:711~720
11 Chen W, Mahadomrongkul V, Berger UV, et al Aoki C and Rosenberg PA, The glutamate transporter GLT1a is expressed in excitatory axon terminals of mature hippocampal neurons. J Neurosci, 2004, 24: 1136~1148
12 Chen W, Aoki C, Mahadomrongkul V, et al Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain. J Neurosci, 2002, 22(6):2142~2152
13 Dirnagl U, Simon RP, Hallenbeck JM. Ischemic tolerance and endogenous neuroprotection. Trends Neurosci, 2003, 26(5):248~254
14 Danbolt, N.C. Glutamate uptake. Prog Neurobiol, 2001, 65:1~105
15 Davis KE, Straff DJ, Weinstein EA, et al Multiple signaling pathways regulate cell surface expression and activity of the excitatory amino acid carrier 1 subtype of Glu transporter in C6 glioma. J Neurosci, 1998, 18:2475~2485
16 Douen AG, Akiyama K, Hogan MJ, et al Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebral cortex plasma membranes. J Neurochem, 2000, 75(2):812~818
17 During MJ, Spencer DD. Extracellular lippocampal glutamate and spontaneous seizure in the conscious human brain. Lancet, 1993, 341:607~1610
18 Euler T, Wassle H. Immunocytochemical identification of cone bipolarcells in the rat retina. J Comp Neurol, 1995, 361:461~478
19 Feustel PJ, Jin Y, Kimelberg HK, Volume-regulated anion channels are the predominant contributors to release of excitatory amino acids in the ischemic cortical penumbra. Stroke, 2004, 35(5):1164~1168
20 Figiel M, Engele J. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a Neuron-Derived Peptide Regulating Glial Glutamate Transport and Metabolism. J Neurosci, 2000, 20(10):3596~3605
21 Figiel M, Maucher T, Rozyczka J, et al Regulation of glial glutamate transporter expression by growth factors. Exp Neurol, 2003, 183:124~135
22 Futura A, Rothstein JD, Martin LJ, Glutamate Transporter Protein Subtypes Are Expressed Differentially during Rat CNS Development. J Neurosci, 1997, 17:8363~8375
23 Garcia L, Burda J, Hrehorovska M, et al Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression. J Neurochem, 2004, 88(1):136~147
24 Gegelashvili G, Civenni G, Racagni G, et al Glutamate receptor agonists up-regulate the glutamate transporter GLAST in astrocytes. Neuroreport, 1996, 8:261~265
25 Gegelashvili G, Danbolt N C, Schousboe A. Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. J Neurochem, 1997, 69:2612~2615
26 Gegelashvili G, Robinson MB, Trotti D, et al Regulation of glutamate transporters in health and disease, Prog Brain Res, 2001, 132:267~286
27 Gegelashvili G, Schousboe A Cellular distribution and kinetic properties of high-affinity glutamate transporters. Brain Res Bull, 1998, 45:233~238
28 Gogas KR, Glutamate-based therapeutic approaches: NR2B receptor antagonists. Curr Opin Pharmacol, 2006, 6(1):68~74.
29 Grabb MC, Lobner D, Turetsky DM, et al Preconditioned resistance to oxygen-glucose deprivation-induced cortical neuronal death: alterations in vesicular GABA and glutamate release. Neuroscience, 2002,115(1):173~183
30 Greenamyre JT, Young AB. Excitatory amino acids and Alzheimer’s disease. Neurobiol. Aging, 1989, 10:593~602
31 Guo H, Lai L, Butchbach ME, et al Increased expression of the glial glutamate transporter EAAT2 modulates excitotoxicity and delays the onset but not the outcome of ALS in mice. Hum Mol Genet, 2003, 12(19):2519~2532
32 Hayashi M, Hayashi R, Tanii H, et al The influence of neuronal cells on the development of glutamine synthetase in astrocytes in vitro. Dev Brain Res, 1988, 41:37~42
33 Heurteaux C, Lauritzen I, Widmann C, et al Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning. PNAS, 1995, 92:4666~4670
34 Hong JS, McGinty JF, Grimes L, et al Seizure-induced alterations in the metabolism of hippocampal opioid peptides suggest opioid modulation of seizure-related behaviors. NIDA Res Monogr, 1988, 82:48~66
35 Hu WH, Walters WM, Xia XM, et al Neuronal glutamate transporter EAAT4 is expressed in astrocytes. Glia, 2003, 44(1):13~25
36 Kalandadze A, Wu Y, Robinson MB. Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486. J Biol Chem, 2002, 29;277(48):45741~45750.
37 Kanai Y, Hediger MA. The glutamate and neutral amino acid transporter family: physiological and pharmacological implications. Eur J Pharmacol, 2003, 479(1-3):237~247
38 Kanai Y, Hediger MA. The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects. Pflugers Arch, 2004, 447(5):469~479
39 Kanai Y, Trotti D, Nussberger S, et al The high affinity glutamate transporter family. In: Neurotransmitter transporters: structure, function, and regulation (Reith MEA, ed), 1997, pp171–213. Totowa, NJ: Humana.
40 Kato H, Kogure K. Biochemical and molecular characteristics of the brain with developing cerebral infarction. Cell Mol Neurobiol, 1999, 19:93~108
41 Katsuki H, Akaike A. Excitotoxic degeneration of hypothalamic orexin neurons in slice culture. Neurobiol Dis, 2004, 15(1):61~69
42 Kosugi T, Kawahara K, Reversed actrocytic GLT-1 during ischemia is crucial to excitotoxic death of neurons, but contributes to the survival of astrocytes themselves, Neurochem Res, 2006, 31(7):933~943
43 Lee J, Lee J E, Cho EH, et al An essential histidine residue in GTP binding domain of bovine brain glutamate dehydrogenase isoproteins. Mol Cells, 2001, 12:121~126
44 Lehre KP, Danbolt NCThe number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci, 1998, 18:8751~8757
45 Lehre K P, Levy LM, Ottersen OP, et al Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci, 1995, 15:1835~1853
46 Levenson J, Endo S, Kategaya LS, et al Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia. Proc Natl Acad Sci USA, 2000, 97:12858~12863
47 Levenson J, Weeber E, Selcher JC, et al Long-term potentiation and contextual fear conditioning increase neuronal glutamate uptake. Nat Neurosci, 2002, 5:155~161
48 Lin CL, Bristol LA, Jin L, et al Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron, 1998, 20(3):589~602
49 Lipton P. Ischemic cell death in brain neurons. Physiol Rev, 1999, 79:1431~1568
50 Liu GJ, Madsen BW. PACAP-38 modulates activity of NMDA receptors in cultured chick cortical neurons. J Neurophysiol, 1997, 78:2231~2234
51 Maleszka R, Helliwell P, Kucharski R. Pharmacological interference with glutamate re-uptake impairs long-term memory in the honeybee, apis mellifera. Behav Brain Res, 2000, 115:49~53
52 Mansouri FA, Motamedi F, Fathollahi Y. Chronic in vivo morphine administration facilitates primed-bursts-induced long-term potentiation of Schaffer collateral-CA1 synapses in hippocampal slices in vitro. Brain Res, 1999, 815:419~423
53 Maragakis NJ, Dykes-Hoberg M, Rothstein JD. Altered expression of the glutamate transporter EAAT2b in neurological disease. Ann Neurol, 2004, 55:469~477
54 Martin J-L, Gasser D, Magistretti PJ. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide potentiate c-fos expression induced by glutamate in cultured cortical neurons. J Neurochem, 1995, 65:1~9.
55 Masliah E, Alford M, DeFeresa R, et al Deficient glutamate transport is associated with neurodegeneration in Alzheimer`s disease. Ann Neurol 1996, 40:759~766
56 Masliah E, Alford M, Mallory M, et al Abnormal glutamate transport function in mutant amyloid precursor protein transgenic mice. Exp Neurol, 2000, 163(2):381~387
57 Mathern GW, Menoza D, Lozada A, et al Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy. Neurology, 1999, 52:453~472
58 Matsumoto Y, Yamamoto S, Suzuki Y, et al. Na+/H+ exchanger inhibitor, SM-20220, is protective against excitotoxicity in cultured cortical neurons. Stroke, 2004, 35(1):185~190
59 Meldrum BS. The role of glutamate in epilepsy and other disorders. [Review] Neurology 1994, 44:S14~23
60 Meldrum BS, Akbar MT, Chapman AG. Glutamate receptors and transporters in genetic and acquired models of epilepsy. [Review] Epilepsy Res, 1999, 36:189~204
61 Mitani A, Tanaka K. Functional Changes of Glial Glutamate Transporter GLT-1 during Ischemia: An In Vivo Study in the Hippocampal CA1 of Normal Mice and Mutant Mice Lacking GLT-1. J Neurosci, 2003, 23(18):7176~7182
62 Munch C, Ebstein M, Seefried U, et al Alternative splicing of the 5'-sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis. J Neurochem, 2002, 82(3):594~603
63 Namura S, Maeno H, Takami S, et al Inhibition of glial glutamate transporter GLT-1 augments brain edema after transient focal cerebral ischemia in mice. Neurosci Lett, 2002, 324(2):117~120
64 Nedergaard M, Takano Y, Hansen AJ. Beyond the role of glutamate as a neurotransmitter. Nat Rev Neurosci, 2002,3(9):748~755
65 Neugebauer V, Glutamate receptor ligands. Handb Exp Pharmacol, 2007, 177:217~249
66 Nicotera P, Bano D. The enemy at the gates. Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell, 2003, 115(7):768~770
67 Nishino K, Nowak Jr TS. Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning. J Cereb Blood Flow Metab. 2004, 24(2):167~178
68 Ng KT, O'Dowd BS, Rickard NS, et al Complex roles of glutamate in the Gibbs-Ng model of one-trial aversive learning in the new-born chick. Neurosci Biobehav Rev, 1997, 21:45~54
69 Olney JW. Excitotoxic amino acids and neuropsychiatric disorders. Annu Rev Pharmacol Toxicol, 1990, 30:47~71
70 O'Shea RD. Roles and regulation of glutamate transporters in the central nervous system. Clin Exp Pharmacol Physiol, 2002, 29(11):1018~1023
71 Payet O, Maurin L, Bonne C, et al Hypoxia stimulates glutamate uptake in whole rat retinal cells in vitro. Neurosci Lett, 2004, 356(2):148~150
72 Pellegri G, Magistretti PJ, Martin J-L. VIP and PACAP potentiate the action of glutamate on BDNF expression in mouse cortical neurones. Eur J Neurosci, 1998, 10:272~280.
73 Perego C, Vanoni C, Bossi M, et al The GLT-1 and GLAST Glutamate Transporters Are Expressed on Morphologically Distinct Astrocytes and Regulated by Neuronal Activity in Primary Hippocampal Cocultures. J Neurochem, 2000, 75(3):1076~1084
74 Perry TL, Hansen S. What excitotoxin kills striatal neurons in Huntington’s disease? Clues from neurochemical studies. Neurology, 1990, 40:20~24
75 Proper EA, Hoogland G, Kappen SM, et al Distribution of glutamate transporters in the hippocampus of patients with pharmaco-resistant temporal lobe epilepsy. Brain, 2002,125:32~43
76 Pu L, Bao GB, Xu NJ, et al Hippocampal long-term potentiation is reduced by chronic morphine treatment and can be restored by re-exposure to opiates. J Neurosci, 2002, 22:1914~1921
77 Rao VL, Dogan A, Todd KG, et al Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain. J Neurosci, 2001a, 21(6):1876~1783
78 Rao VL, Dogan A, Bowen KK, et al Antisense knockdown of the glial glutamate transporter GLT-1 exacerbates hippocampal neuronal damage following traumatic injury to rat brain. Eur J Neurosci, 2001b, 13(1):119~128
79 Rauen T, Kanner BI. Localization of the glutamate transporter GLT-1 in rat and macaque monkey retinae. Neurosci Lett, 1994, 169:137~140
80 Rauen T. Diversity of glutamate transporter expression and function in the mammalian retina. Amino Acids, 2000, 19:53~62
81 Raval AP, Dave KR, Mochly-Rosen D, et al Epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice. J Neurosci, 2003,23(2):384~391
82 Reye P, Sullivan R, Fletcher EL, et al Distribution of two splice variants of the glutamate transporter GLT1 in the retinas of humans, monkeys, rabbits, rats, cats, and chickens. J Comp Neurol, 2002a, 445:1~12
83 Reye P, Sullivan R, Pow DV. Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina. J Comp Neurol, 2002b, 447:323~330
84 Rintoul GL, Filiano AJ, Brocard JB, et al Glutamate decreases mitochondrial size and movement in primary forebrain neurons. J Neurosci, 2003, 23(21):7881~7888
85 Robinson MB, Dowd LA. Heterogeneity and functional properties of subtypes of sodium-dependent glutamate transporters in the mammalian central nervous system. Adv Pharmacol, 1997, 37:69~115
86 Romera C, Hurtado O, Botella SH, et al In vitro ischemic tolerance involves upregulation of glutamate transport partly mediated by the TACE/ADAM17-tumor necrosis factor-alpha pathway. J Neurosci, 2004, 24(6):1350~1357
87 Rothstein JD, Dykes-Hoberg M, Pardo CA, et al Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron, 1996, 16:675~686
88 Rothstein JD, Patel S, Regan MR, et al -Lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature, 2005, 433:73~77
89 Rothstein JD, Van Kammen M, Levy AI, et al Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann Neurol, 1995, 38:73~84
90 Rozyczka J, Figiel M, Engele J. Endothelins negatively regulate glial glutamate transporter expression. Brain Pathol, 2004, 14(4):406~414
91 Sanchez-Gomez MV, Alberdi E, Ibarretxe G, et al Caspase-dependent and caspase-independent oligodendrocyte death mediated by AMPA and kainate receptors. Neurosci, 2003, 23(29):9519~9528
92 Schlag BD, Vondrasek JR, Munir M, et al Regulation of glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons. Mol Pharmacol, 1998, 53:355~369
93 Shigery Y, Seal RP, Shimamoto K. Molecular pharmacology of glutamate transporters, EAATs and VGLUTs. Brain Res Brain Res Rev, 2004, 45(3):250~265
94 Shobha K, Vijayalakshmi K, Alladi PA, et al Altered in-vitro and in-vivo expression of glial glutamate transporter-1 following exposure to cerebrospinal fluid of amyotrophic lateral sclerosis patients. J Neurol Sci, 2007, 254(1-2):9~16.
95 Sonnewald U, Westergaard N, Schousboe A. Glutamate transport and metabolism in astrocytes. Glia, 1997, 21:56~63
96 Stella N, Magistretti PJ. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) potentiate the glutamate-evoked release of arachidonic acid from mouse cortical neurons. Evidence for a cAMP-independent mechanism. J Biol Chem, 1996, 271:23705~23710
97 Stoffel W, Korner R, Wachtmann D, et al Functional analysis of glutamate transporters in excitatory synaptic transmission of GLAST1 and GLAST1/EAAC1 deficient mice. Mol Brain Res, 2004, 128:170~181
98 Suchak SK, Baloyianni NV, Perkinton MS, et al The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings. J Neurochem, 2003, 84(3):522~32
99 Sullivan R, Rauen T, Fischer F, et al Cloning, transport properties, and differential localization of two splice variants of GLT-1 in the rat CNS: Implications for CNS glutamate homeostasis. Glia, 2004, 45(2):155~169
100 Sun Y, Jin K, Peel A, et al Greenberg DA. Neuroglobin protects the brain from experimental stroke in vivo. PNAS, 2003; 100(6):3497~3500
101 Swanson RA, Liu J, Miller JW, et al Neuronal regulation of glutamate transporter subtype expression in astrocytes. J Neurosci, 1997, 17:932~940
102 Szatkowski M, Attwell D. Triggering and execution of neuronal death in brain ischemia: Two phases of glutamate release by different mechanisms. Trends Neurosci, 1994, 17:359~365
103 Takagi K, Ginsberg MD, Globus MY, et al Effect of hyperthermia on glutamate release in ischemic penumbra after middle cerebral artery occlusion in rats. Am J Physiol, 1994, 267:H1770~1776
104 Tanaka K, Watase K, Manabe T, et al Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science, 1997, 276:1699~1702
105 Tanaka K. Functions of glutamate transporters in the brain. Neurosci Res, 2000, 37:15~19
106 Tauskela JS, Brunette E, Monette R, et al Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling. Am J Physiol Cell Physiol, 2003, 285(4):C899~911
107 Tessler S, Danbolt NC, Faull RL, et al Expression of the glutamate transporters in human temporal lobe spilepsy. Neurossience, 1999, 88:1083~1091
108 Thorlin T, Roginski RS, Choudhury K, et al Regulation of the glial glutamate transporter GLT-1 by glutamate and delta-opioid receptor stimulation. FEBS Lett, 1998, 425:453~459
109 Uchiyama-Tsuyuki Y, Araki H, Yae T, et al Changes in the extracellular concentrations of amino acids in the rat striatum during transient focal cerebral ischemia. J Neurochem, 1994, 62:1074~1078
110 Ullensvang K, Lehre K P, Storm-Mathisen J, et al Differential developmental expression of the two rat brain glutamate transporter proteins GLAST and GLT. Eur J Neurosci, 1997, 9:1646~1655
111 Van Damme P, Dewil M, Robberecht W, et al Excitotoxicity and amyotrophic lateral sclerosis. Neurodegener Dis, 2005, 2(3-4):147~159
112 Van den Bosch L, The causes and mechanism of selective motor neuron death in amyotrophic lateral sclerosis. Werh K Acad Geneeskd Belg,2006, 68(4):249~269
113 Velly LJ, Guillet BA, Masmejean FM, et al Neuroprotective effects of propofol in a model of ischemic cortical cell cultures: role of glutamate and its transporters. Anesthesiology, 2003, 99(2):368~375
114 Watase K, Hashimoto K, Kano M, et al Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. Eur J Neurosci, 1998, 10:976~988
115 Whetsell WO Jr J. Current concepts of excitotoxicity. Neuropathol Exp Neurol, 1996, 55(1):1~13
116 Whetsell Jr WO, Shapira NA. Neuroexcitation, excitotoxicity, and human neurological disease. Lab Invest, 1993, 68:372~387
117 Williams JT, Christie MJ, Manzoni O. Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev, 2001, 81:99~343
118 Wilson CL, Maidment NT, Shomer MH, et al Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy. Epilepsy Res, 1996, 265:245~254
119 Won MH, Kang T-C, Jeon GS, et al Immunohistochemical detection of oxidative DNA damage induced by ischemia-reperfusion insults in gerbil hippocampus in vivo. Brain Res, 1999, 836:70~78
120 Won MH, Kang TC, Park SK, et al The alterations of N-Methyl-D-aspartate receptor expressions and oxidative DNA damage in the CA1 area at the early time after ischemia-reperfusion insult. Neurosci Lett, 2001, 301:139~142
121 Xu NJ, Bao L, Fan HP, et al Morphine Withdrawal Increases Glutamate Uptake and Surface Expression of Glutamate Transporter GLT1 at Hippocampal Synapses. J Neurosci, 2003, 23(11):4775~4784
122 Yamada K, Watanabe M, Shibata T, et al Glutamate transporter GLT-1 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression. J Neurosci, 1998, 18:5706~5713
123 Young KC, McGehee DS, Brorson JR. Glutamate receptor expression andchronic glutamate toxicity in rat motor cortex. 2007, Epub ahead of print
124 Zacco A, Togo J, Spence K, et al 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitors protect cortical neurons from excitotoxicity. Neuroscience, 2003, 23(35):11104~11111
125 Zelenaia O, Schlag BD, Gochenauer GE, et al Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB. Mol Pharmacol, 2000, 57:667~678
126 Zhou AM, Li WB, Li QJ, et al A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res, 2004, 48(4):379~404